Penzes Z, Tibbles K, Shaw K, Britton P, Brown T D, Cavanagh D
Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, United Kingdom.
Virology. 1994 Sep;203(2):286-93. doi: 10.1006/viro.1994.1486.
The Beaudette strain of IBV was passaged 16 times in chick kidney cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage 6. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12,423 to 18,744 in the IBV genome, and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.
传染性支气管炎病毒（IBV）的博德特毒株在鸡肾细胞中传代16次。通过Northern杂交分析总细胞RNA，并用对应于基因组5'端前2kb（不包括前导序列）以及基因组3'端后1.8kb的32P标记的cDNA探针进行探测。在传代6次时检测到一种新的、有缺陷的IBV RNA种类（CD - 91）。存在于总细胞提取物RNA和传代15的寡聚（dT）30选择的RNA中的缺陷RNA，通过逆转录聚合酶链反应（RT - PCR）扩增得到四个片段。所使用的寡核苷酸经过挑选，使得仅能扩增CD - 91 RNA而非基因组RNA。PCR产物的克隆和测序表明，CD - 91由9.1kb组成，具有基因组的三个区域。它包含来自基因组5'端的1133个核苷酸、来自基因1b的6322个核苷酸（对应于IBV基因组中第12423至18744位）以及来自基因组3'端的1626个核苷酸。在第749位，CD - 91 RNA缺失一个核苷酸，即腺嘌呤残基。通过Northern杂交检测到，病毒粒子中CD - 91 RNA的含量高于亚基因组mRNA。
