Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments

• Published in: Avian diseases 1999-04, Vol.43 (2), p.219-226
• Main Authors: Todd, D, Weston, J.H, Mawhinney, K.A, Laird, C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Avian encephalomyelitis virus; Base Sequence; characterization; Chick Embryo; Chromosome Mapping - veterinary; cloning; Cloning, Molecular; Complementary DNA; DNA; DNA, Complementary - chemistry; DNA, Viral - chemistry; Encephalomyelitis Virus, Avian - genetics; Enterovirus Infections - diagnosis; Enterovirus Infections - virology; genome; Genomes; Microscopy, Electron - veterinary; Molecular Sequence Data; nucleotide sequences; Open reading frames; Picornaviridae; polymerase chain reaction; Polymerase Chain Reaction - veterinary; Reverse transcriptase polymerase chain reaction; RNA; RNA probes; RNA, Viral - chemistry; Viruses
• ISSN: 0005-2086; 1938-4351
• DOI: 10.2307/1592611

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.