Duchemin Jean-Bernard, Paradkar Prasad N
CSIRO Health and Biosecurity, Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Victoria, 3220, Australia.
Virol J. 2017 Jun 5;14(1):103. doi: 10.1186/s12985-017-0770-0.
Mosquitoes are responsible for transmission of viruses, including dengue, West Nile and chikungunya viruses. Female mosquitoes are infected when they blood-feed on vertebrates, a required step for oogenesis. During this process, mosquitoes encounter high iron loads. Since iron is an essential nutrient for most organisms, including pathogens, one of the defense mechanisms for the host includes sequestration of iron away from the invading pathogen. Here, we determine whether iron availability affects viral replication in mosquitoes.
To elucidate effect of iron availability on mosquito cells during infection, Culex cells were treated with either ferric ammonium citrate (FAC) or the iron chelator, deferoxamine (DFX). Real time RT-PCR was performed using ferritin (heavy chain) and NRAMP as a measure of iron homeostasis in cells. To determine iron requirement for viral replication, Culex cells were knocked down for NRAMP using dsRNA. Finally, the results were validated in Culex mosquito-infection model, by treating infected mosquitoes with DFX to reduce iron levels.
Our results show that infection of Culex cells led to induction in levels of ferritin (heavy chain) and NRAMP mRNAs in time-dependent manner. Results also showed that treatment of cells with FAC, reduced expression of NRAMP (iron transporter) and increase levels of ferritin (heavy chain). Interestingly, increasing iron levels increased viral titers; while reducing intracellular iron levels, either by NRAMP knock-down or using DFX, reduced viral titers. The results from Culex mosquito infection showed that mosquitoes treated with DFX had reduced viral titers compared with untreated controls in midgut as well as carcass 8 days pi. Saliva from mosquitoes treated with DFX also showed reduced viral titers compared with untreated controls, indicating low viral transmission capacity.
Our results indicate that iron is required for viral replication in mosquito cells. Mosquitoes respond to viral infection, by inducing expression of heavy chain ferritin, which sequesters available iron, reducing its availability to virus infected cells. The data indicates that heavy chain ferritin may be part of an immune mechanism of mosquitoes in response to viral infections.
蚊子是包括登革热、西尼罗河病毒和基孔肯雅病毒在内的多种病毒的传播媒介。雌蚊在吸食脊椎动物血液时会被感染，这是卵子发生的必要步骤。在此过程中，蚊子会接触到高负荷的铁。由于铁是包括病原体在内的大多数生物的必需营养素，宿主的一种防御机制包括将铁隔离，使其远离入侵的病原体。在此，我们确定铁的可利用性是否会影响蚊子体内的病毒复制。
为了阐明感染期间铁的可利用性对蚊子细胞的影响，用柠檬酸铁铵（FAC）或铁螯合剂去铁胺（DFX）处理库蚊细胞。使用铁蛋白（重链）和天然抗性相关巨噬蛋白（NRAMP）进行实时逆转录聚合酶链反应，以衡量细胞内铁稳态。为了确定病毒复制对铁的需求，使用双链RNA敲低库蚊细胞中的NRAMP。最后，通过用DFX处理受感染的蚊子以降低铁水平，在库蚊感染模型中验证结果。
我们的结果表明，库蚊细胞感染会导致铁蛋白（重链）和NRAMP mRNA水平呈时间依赖性诱导。结果还表明，用FAC处理细胞会降低NRAMP（铁转运蛋白）的表达，并增加铁蛋白（重链）的水平。有趣的是，铁水平的增加会提高病毒滴度；而通过敲低NRAMP或使用DFX降低细胞内铁水平，则会降低病毒滴度。库蚊感染的结果表明，与未处理的对照组相比，用DFX处理的蚊子在感染后8天，中肠和胴体中的病毒滴度降低。与未处理的对照组相比，用DFX处理的蚊子的唾液中病毒滴度也降低，表明病毒传播能力较低。
我们的结果表明，铁是蚊子细胞中病毒复制所必需的。蚊子通过诱导重链铁蛋白的表达来应对病毒感染，重链铁蛋白会隔离可用铁，从而减少其对病毒感染细胞的可用性。数据表明，重链铁蛋白可能是蚊子应对病毒感染的免疫机制的一部分。
