Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking

The symbiont Wolbachia limits viral replication in Aedes aegypti and significantly reduces dengue fever incidence in humans following field release. Despite plans for its widespread use, Wolbachia’s mode of action remains poorly understood. Many studies suggest that the mechanism of viral blocking is likely multifaceted, involving aspects of immunity, cellular stress, and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined the expression of alpha-Mann-2a in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference (RNAi), affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue and time dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the Togavirus chikungunya (CHIKV), indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.共生体沃尔巴克氏体（Wolbachia）可抑制埃及伊蚊（Aedes aegypti）体内的病毒复制，并在野外释放后显著降低人群中的登革热发病率。尽管已有大规模应用该策略的规划，但沃尔巴克氏体发挥作用的具体分子机制仍未被充分阐明。多项研究表明，病毒阻断的机制可能具有多面性，涉及免疫应答、细胞应激以及营养竞争等多个层面。本团队此前的一项研究通过人工筛选技术，鉴定出了一个与病毒阻断相关的伊蚊候选基因——α-甘露糖苷酶-2a（alpha-mannosidase-2a, α-Mann-2a），该基因被预测参与蛋白质糖基化（protein glycosylation）过程。蛋白质糖基化通路通常参与复杂的宿主-病毒互作过程，但目前尚未有关于α-甘露糖苷酶在伊蚊-病毒互作中发挥功能的相关报道。 本研究检测了α-Mann-2a在病毒和沃尔巴克氏体感染后的表达模式，并探究了通过RNA干扰（RNA interference, RNAi）抑制该基因表达后是否会影响病毒载量。研究结果显示，登革病毒（Dengue virus, DENV）感染会以组织特异性和时间依赖性的方式调控α-Mann-2a的表达，而沃尔巴克氏体感染则不会对该基因的表达产生影响。在无沃尔巴克氏体感染的伊蚊中，敲低α-Mann-2a的表达后，中肠内的登革病毒感染率显著升高，提示α-Mann-2a可对病毒感染产生干预作用。对披膜病毒科的基孔肯雅病毒（chikungunya, CHIKV）进行基因敲低后也得到了一致的结果，表明α-Mann-2a在伊蚊中肠内可能具有广谱的抗病毒活性。有趣的是，在携带沃尔巴克氏体的伊蚊中，我们无法成功敲低α-Mann-2a的表达。 本研究还提供了证据，表明α-Mann-2a可能调控另一个被预测参与病毒阻断和细胞黏附过程的基因——钙粘蛋白87a（cadherin87a）的转录水平。上述研究结果支持这一假说，即糖基化通路与细胞黏附通路可能广泛参与埃及伊蚊体内的病毒感染过程。