Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments.
cDNA fragments were generated from RNA extracted from preparations of avian encephalomyelitis virus (AEV) by a reverse transcription-polymerase chain reaction (RT-PCR) strategy, which exploited the probability that AEV is a picornavirus. Rapid amplification of the 3' cDNA ends, which utilized an oligo d(T)-based primer that hybrizes to the putative Poly (A) tract at the 3' terminus of picornavirus RNA, produced a 3.8-kbp fragment (3.8-kbp 3' RACE fragment), from which a 2.5-kbp cDNA fragment specific to the extreme 3' terminal region of the AEV genome was cloned. Positive hybridization reactions between RNA from gradient-purified virus and radiolabeled probes confirmed that the cloned 2.5-kbp fragment was AEV specific. The success of the RT-PCR amplification strategy adopted and the results of northern blotting hybridization experiments indicated that the AEV genome is a polyadenylated, single-stranded RNA, approximately 7.5 kb in size. Sequence analysis of a 869-base region at the 3' terminal of the genome indicated that this region encoded a protein with close homologies to picornaviral RNA polymerase proteins. On the basis that the highest levels of protein homologies were observed with hepatitis A virus, it is likely that AEV will be reassigned to a genus other than the enterovirus genus within the virus family Picornaviridae. The AEV-specific cloned DNA fragments and nucleotide sequence information resulting from this investigation may facilitate the development of in situ hybridization and RT-PCR methods that will be useful in AEV diagnosis.
Department of Agriculture for Northern Ireland, Queen's University of Belfast, Stormont, United Kingdom.
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D
Todd
J H
JH
Weston
K A
KA
Mawhinney
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C
Laird
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002874:Chromosome Mapping / Q000662:veterinary; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry*; D001350:Encephalomyelitis Virus, Avian / Q000235:genetics*; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D008854:Microscopy, Electron / Q000662:veterinary; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012367:RNA, Viral / Q000737:chemistry
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral