Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host

To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

迄今为止，禽甲型疱疹病毒（avian alphaherpesvirus）中的传染性喉气管炎病毒（infectious laryngotracheitis virus, ILTV）不同毒株间的重组，此前仅能通过全基因组测序与序列分析在野外样本中检出。既往研究表明，自然重组在ILTV中广泛存在，且证实两株致弱ILTV疫苗毒株间的重组可产生强毒力病毒，在澳大利亚的家禽群中引发大范围疫病。为深入解析ILTV的重组机制，本研究开发了一种TaqMan单核苷酸多态性（single nucleotide polymorphism, SNP）基因分型检测方法，用于在实验条件下检测两株ILTV野外毒株（CSW-1与V1-99 ILTV）间的重组事件。将这两株ILTV毒株共同接种于无特定病原体（specific pathogen free, SPF）鸡体内后，对回收的病毒进行空斑纯化，并采用该SNP基因分型检测方法进行分析。该检测方法在所有共同接种的鸡只体内均检出了ILTV重组体。总计87株回收病毒中有64株（74%）为重组病毒，且检测到23种不同的重组模式，其中部分模式的出现频率更高。本研究结果证实，TaqMan SNP基因分型检测方法是研究ILTV重组的有效工具，同时也表明在SPF鸡体内进行ILTV实验性共同感染时，重组事件频繁发生。若将该工具用于评估自然宿主中的ILTV重组情况，有望极大增进我们对甲型疱疹病毒重组机制的认知。