Synchronization of in vivo MacoNPV-A baculovirus infection by analysis of individual Mamestra configurata larval guts

Many studies have examined the gene expression of baculoviruses during host infection by infecting cultured insect host cells; however, only a few have attempted to characterize the interaction between baculoviruses and insect larvae, which more accurately models the virus-insect relationship. The greatest challenge in assessing gene expression profiles in vivo in the larval gut is the lack of infection synchronization compared to cultured cells. Working with bertha armyworm, Mamestra configurata, larvae infected with the baculovirus Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), viral gene expression was measured using droplet-digital PCR showing that the rate of infection in individual insects varies extremely widely. Subsequent RNA-seq of individual guts revealed that gene expression patterns were consistent in individuals with similar levels of viral gene expression. Therefore, the best approach for analyzing baculovirus gene expression in vivo is to use the expression of an early viral gene as a means to select individuals that are closely matched in infection progress to produce the most synchronized infection cohorts for measuring gene expression profiles. In this way, a profile of gene expression was identified very early in infection that would have been masked by the activity of a few very robustly infected individuals present in sample pools made up of multiple individuals.已有诸多研究通过感染体外培养的昆虫宿主细胞，探究了杆状病毒（baculovirus）在宿主感染过程中的基因表达情况；然而，仅有少数研究尝试阐释杆状病毒与昆虫幼虫之间的互作关系——这一模型更精准地复刻了病毒与昆虫的天然侵染关联。相较于体外培养的细胞，在昆虫幼虫肠道内开展体内基因表达谱分析时，面临的最大挑战在于难以实现侵染过程的同步化。本研究以感染了甘蓝夜蛾核型多角体病毒-A（Mamestra configurata nucleopolyhedrovirus-A，MacoNPV-A）的贝夜蛾（Mamestra configurata）幼虫为研究对象，通过液滴数字PCR（droplet-digital PCR）检测病毒基因表达量，结果显示单头昆虫的侵染速率差异极大。后续对单头幼虫肠道的转录组测序（RNA-seq）分析表明，病毒基因表达水平相近的个体，其基因表达模式具有一致性。因此，若要开展体内杆状病毒基因表达分析，最优方案是借助早期病毒基因的表达量作为筛选标准，挑选侵染进程高度匹配的个体，以构建同步化程度最高的侵染队列，进而精准测定基因表达谱。通过该策略，我们成功鉴定到侵染早期的基因表达谱——若采用多份个体混合的样本池进行分析，这类表达谱会被少量侵染程度极高的个体的基因活性所掩盖。