Division of Biology, Kansas State University, Manhattan, Kansas, USA.
Department of Biological Sciences, Auburn University, Auburn, Alabama, USA.
J Virol. 2021 Jun 10;95(13):e0013621. doi: 10.1128/JVI.00136-21.
Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.
虫媒病毒通过特定的媒介传播，而这种特异性的原因尚不完全清楚。一个促成因素是媒介体内存在组织屏障，如中肠逃逸屏障。我们使用微小 RNA（miRNA）靶向辛德毕斯病毒（SINV）来研究中肠细胞内的复制如何有助于克服埃及伊蚊中的这种屏障。设计了 SINV 构建体，通过将针对中肠特异性 miRNA 的结合位点插入 SINV 基因组的 3'非翻译区（MRE3'miRT）或结构开放阅读框（MRE-ORFmiRT），特异性地在中肠细胞中减弱。在存在这些 miRNA 的情况下，两种 miRNA 靶向病毒的复制效率均低于对照病毒。当蚊子摄入含有 miRNA 靶向病毒的传染性血液餐时，只有约 20%（MRE3'miRT）或 40%（MRE-ORFmiRT）的蚊子发生了传播性感染。相比之下，用对照病毒感染的蚊子几乎都发生了传播。对来自个体蚊子的病毒种群进行深度测序排除了选择插入靶序列中的突变作为这些蚊子传播的原因。在感染了 miRNA 靶向病毒的蚊子中，中肠和尸体中的病毒滴度与感染对照病毒的蚊子相当，没有证据表明传播需要阈值滴度。相反，似乎如果感染在中肠中成功建立，复制和传播在很大程度上是正常的。我们的结果支持这样的假设，即复制是 SINV 克服中肠逃逸屏障的重要因素，但暗示其他因素也可能参与其中。当蚊子在血液餐中摄入虫媒病毒时，病毒必须从中肠逃逸并感染唾液腺才能传播给新宿主。我们使用组织特异性 miRNA 靶向来研究 Sindbis 病毒（SINV）在中肠上皮中复制以在埃及伊蚊中引起传播性感染的要求。我们的结果表明，特异性降低 SINV 在蚊子中的复制能力会降低其在蚊子中建立感染的整体能力，但如果感染建立，复制和传播会正常发生。这些结果与复制在中肠上皮中的重要性一致，有助于虫媒病毒穿过中肠屏障。
