Schnitzlein W M, Winans R, Ellsworth S, Tripathy D N
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801, USA.
Virology. 1995 Jun 1;209(2):304-14. doi: 10.1006/viro.1995.1262.
Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.
目前用于禽呼吸道疾病传染性喉气管炎的疫苗由致病因子——传染性喉气管炎疱疹病毒（ILTV）的自然减毒株组成。由于这些病毒会从接种疫苗的鸡中传播，以及它们可能回复为更具致病性的形式，因此正在考虑使用缺乏毒力因子但保留抗原性的基因工程病毒疫苗。由于疱疹病毒的胸苷激酶（TK）活性与毒力有关，ILTV基因组中编码基因的失活应会使病毒减毒。此外，类似于其他TK缺陷型疱疹病毒，这种ILTV突变体在鸡中诱导保护性反应的能力不应受到损害。因此，尝试对ILTV进行有意的基因改造。为了防止回复突变并能够鉴定修饰后的病毒，通过同源重组在ILTV TK基因内的两个位点之一插入了一个“标记”转录单位（与SV-40 3'-聚腺苷酸化信号序列融合并由伪狂犬病病毒gX基因启动子调控的大肠杆菌lacZ基因）。根据重组病毒产生β-半乳糖苷酶的能力对其进行鉴定和噬斑纯化。通过Southern杂交验证了重组ILTV基因组中预测位点处外源DNA的保留情况。由于它们的复制不受胸腺嘧啶类似物1-(2-氟-2-脱氧-β-D-阿拉伯呋喃糖基)-5-甲基尿嘧啶的影响，这些重组体似乎具有TK缺陷型表型。尽管存在这种明显的缺陷，但将任何一种重组病毒预先接种到鸡中都能使鸡免受强毒ILTV的致死性攻击。此外，与接受亲本病毒的鸡的严重反应相比，接种重组体的鸡的呼吸窘迫程度相对较轻。因此，ILTV可以在不伴随免疫原性丧失的情况下进行基因减毒。
