Scholz E, Welniak E, Nyholm T, Guo P
Department of Veterinary Pathobiology, Purdue Cancer Center, Purdue University, West Lafayette, IN 47907.
J Virol Methods. 1993 Aug;43(3):273-86. doi: 10.1016/0166-0934(93)90146-i.
Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly infectious upper respiratory tract disease in chickens. Vaccine development and basic studies on ILTV have been hampered by the lack of a cell line for the cultivation of this herpesvirus which was identified in 1930. Four different avian cell lines were tested for their suitability to propagate ILTV. Here we report the successful growth of ILTV with a chemically-induced avian hepatoma cell line, while retrovirus transformed cell lines derived from permissive primary cells, were found to be non-permissive for ILTV. After multiple passaging of ILTV in the hepatoma cells, the virus could be grown up to a titre of 1 x 10(7) EID50 per ml with a replication cycle comparable to that in primary hepatocytes. Methods of plaque assay, DNA-transfection, and expression of a reporter gene were established. The gene coding for the bacterial beta-galactosidase gene under the control of the cytomegalovirus (CMV) immediate-early promotor was transiently expressed, indicating that a mammalian herpesvirus promotor was recognized by this avian cell line. Infectious ILTV virions were produced after transfecting this cell-line with purified ILTV DNA. The results indicated that the cell line is suitable for the construction of recombinant ILTV and for the molecular biological study of this important avian pathogen.
传染性喉气管炎病毒（ILTV）是鸡高度传染性上呼吸道疾病的病原体。1930年发现的这种疱疹病毒，由于缺乏用于培养的细胞系，ILTV的疫苗开发和基础研究受到了阻碍。测试了四种不同的禽细胞系对ILTV增殖的适用性。在此我们报告，用化学诱导的禽肝癌细胞系成功培养出了ILTV，而源自允许性原代细胞的逆转录病毒转化细胞系对ILTV不敏感。ILTV在肝癌细胞中多次传代后，病毒滴度可达每毫升1×10⁷ EID₅₀，其复制周期与在原代肝细胞中的相当。建立了噬斑测定、DNA转染和报告基因表达的方法。在巨细胞病毒（CMV）立即早期启动子控制下编码细菌β-半乳糖苷酶基因的基因被瞬时表达，表明该禽细胞系能识别哺乳动物疱疹病毒启动子。用纯化的ILTV DNA转染该细胞系后产生了有传染性的ILTV病毒粒子。结果表明，该细胞系适用于构建重组ILTV以及对这种重要禽病原体进行分子生物学研究。
