Infectious Diseases of Poverty. 2022.
DOI : https://doi.org/10.1186/s40249-022-00990-0
公众号“BMC科研永不止步”：
https://mp.weixin.qq.com/s/nNNndRa6PsCwrDH4Kw0gxQ
BMC Blog Network：
https://blogs.biomedcentral.com/bugbitten/2022/09/30/armigeres-subalbatus-a-potential-vector-for-zika-virus/
中国科学报：
https://www.smu.edu.cn/rdyjs/info/1009/1281.htm
https://news.sciencenet.cn/sbhtmlnews/2022/10/371487.shtm?id=371487
寨卡病毒（Zika virus, ZIKV）是一种主要由媒介伊蚊（埃及伊蚊和白纹伊蚊等）传播的黄病毒。寨卡病毒感染可导致人类先天性小头畸形和格林-巴利综合征等病患。寨卡病毒于1947年从乌干达寨卡森林的一只发热恒河猴体内被发现，后来在同一森林的非洲伊蚊中被分离。2007年，太平洋西部加罗林群岛中的雅浦岛发生了寨卡病毒的第一次暴发。2013年，法属波利尼西亚发生了更大规模的寨卡病毒流行。2015 年，巴西寨卡病毒暴发流行并导致小头症病例激增；截至 2016 年初，该流行病已迅速扩散到美洲、亚洲等30多个国家和地区。2016年2月1日，世界卫生组织将寨卡病毒及小头症列为“国际关注的突发公共卫生事件”（PHEIC）。
骚扰阿蚊隶属于库蚊亚科，在形态学和遗传系统发生分类上更接近伊蚊属蚊虫。骚扰阿蚊兼吸人血和畜血，可传播多种病原体，包括寄生虫和病毒，如丝虫和日本脑炎病毒。幼虫孳生地类型包括竹筒、树洞、稀粪池、污水坑、下水道和容器中的积水。已有学者相继从中国贵州省和泰国等地野外捕获的骚扰阿蚊样本中检测和分离出寨卡病毒，这些发现引起了人们对骚扰阿蚊是否可传播寨卡病毒的担忧。寨卡病毒可以通过患者尿液排出体外，被寨卡病毒污染的水体可能是媒介伊蚊传播该病毒的一个途径。由于骚扰阿蚊喜好的孳生水体更容易被患者排泄物污染，因此骚扰阿蚊对于寨卡病毒易感性和传播性的研究显得尤为重要。
本研究首先从中国广东省广州市的一个大学校园（23°12'03.1''N，113°17'20.1''E）采集了一株骚扰阿蚊，并通过形态学和细胞色素氧化酶亚基1线粒体基因COI测序对该物种进行了鉴定。将寨卡病毒上清与去纤维化羊血混合后喂饲骚扰阿蚊成蚊。血餐后第4天，中肠组织中寨卡病毒检测阳性率为18%，表明了寨卡病毒可以感染骚扰阿蚊中肠。血餐后第7天，卵巢组织的阳性率为8.3%。血餐后第10天，唾液腺阳性率为4.2%。这些结果表明寨卡病毒可以感染并突破中肠和唾液腺屏障。感染成蚊分泌的唾液中也检测到了寨卡病毒，这提示骚扰阿蚊具有将寨卡病毒传播给宿主的潜在风险。
将感染寨卡病毒的骚扰阿蚊雌蚊叮咬4日龄乳鼠。饲养8天后，在乳鼠脑组织中运用RT-PCR和空斑实验均检测到寨卡病毒，进一步证明骚扰阿蚊可以通过叮咬将寨卡病毒传播给宿主。
在骚扰阿蚊幼虫孳生水体中添加含寨卡病毒的人工尿液，随后在四龄幼虫体内检测到寨卡病毒。感染个体羽化后至成蚊阶段后，在成蚊唾液腺中同样可检测到寨卡病毒。这说明，与伊蚊一样，骚扰阿蚊也可在寨卡病毒尿液污染的水体中被感染。
为了进一步确认寨卡病毒在幼虫体内的分布，作者将感染的幼虫制成切片并通过免疫组织化学显色定位寨卡病毒。结果在中肠、肛乳头和其他组织中都观察到病毒颗粒。这表明寨卡病毒可以通过中肠和肛乳头感染骚扰阿蚊幼虫。
用相同的方法，对成蚊喂饲含有登革2型病毒的血餐、在幼虫孳生水体中添加含有登革病毒的人工尿液，结果均未在蚊虫体内检测到登革病毒。综上，作者得出结论，从中国广东分离出的一株骚扰阿蚊，在实验室条件下可以感染和传播寨卡病毒，但不能感染和传播登革2型病毒。此外，骚扰阿蚊幼虫也可能在含有寨卡病毒尿液污染的水体中被感染。本研究探究了骚扰阿蚊对寨卡病毒和登革病毒的媒介能力，揭示了骚扰阿蚊是一种寨卡病毒的潜在传播媒介，为寨卡病毒等蚊媒传染病的防控提供了理论和实验证据。
Zika virus (ZIKV) is a flavivirus mainly transmitted by Aedes mosquitoes, namely Aedes aegypti and Aedes albopictus. Zika virus infection can lead to human illness with congenital microcephaly and post-infective neurological syndromes, particularly Guillain-Barré syndrome. ZIKV was first isolated in 1947 from a febrile rhesus macaque monkey in the Zika Forest of Uganda and later isolated in Aedes africanus mosquitoes from the same forest. The first known ZIKV outbreak occurred on the isolated island of Yap in 2007, and a larger epidemic occurred in French Polynesia. In 2015, a severe Zika outbreak occurred in Brazil with increased cases of microcephaly, the epidemic rapidly spread to more than 30 other countries. WHO classified ZIKV as a “public health emergency of international concern” (PHEIC) in 2016.
Armigeres subalbatus belongs to the Culicinae subfamily, and is considered closer in morphology and genetic phylogeny to Aedes mosquitoes. Ar. subalbatus sucks both human and animal blood and is a vector of multiple pathogens, including parasites and viruses, such as Filaria and Japanese encephalitis virus. Larvae mainly develop in bamboo tubes, tree holes, dilute cesspools, sewage pits, sewers, and standing water in containers. Several investigations report that ZIKV has been detected in field-caught Ar. subalbatus, in Guizhou Province, China, and Thailand. These findings raised concerns that Ar. subalbatus may act as a vector of ZIKV. Since ZIKV can be excreted through urine, water containing ZIKV can be a transmission route of ZIKV. The rearing water of Ar. subalbatus larvae are more likely to be contaminated by the excrement of patients during the epidemic.
In our study, Ar. subalbatus was collected from a campus (23°12′03.1′′N, 113°17′20.1′′E) located in Guangzhou City, Guangdong Province, China. Representative morphology and the cytochrome coxidase subunit 1 mitochondrial gene (COI) sequencing confirmed the identity of Ar. subalbatus. The mosquitoes were maintained at the laboratory for a series of experiments. Mosquito adults were allowed to feed on the defibrinated sheep blood meal mixed with ZIKV supernatant. On day four after the blood meal, ZIKV was detected in the midgut tissue with a positive rate of 18%. The results indicated that ZIKV could infect the midguts of Ar. subalbatus. On day seven after the blood meal, ZIKV could be detected in ovaries with a positive rate of 8.3%, and on day 10, the positive rate of saliva was 4.2%. These findings suggest that ZIKV can infect and overcome the midgut and salivary gland barriers of Ar. subalbatus. In addition, ZIKV can be detected in saliva samples collected from ZIKV-positive mosquitoes, which indicated that Ar. subalbatus has the potential risk of transmitting ZIKV to host organisms.
The ZIKV-positive female adults were allowed to bite 4-day-old suckling mice. The RT-PCR and plaque assay showed that ZIKV could be detected in brain tissue of those bitten mice. These data demonstrated that the infected Ar. subalbatus can transmit ZIKV to suckling mice by bite.
When ZIKV was added daily to the rearing water, ZIKV could be detected in fourth instar larvae, and even the virus could spread to the salivary gland of grew-up adults. As with the Aedes mosquito, larvae of Ar. subalbatus can be infected by ZIKV in an artificial urine infection environment.
To further confirm the distribution of ZIKV in larvae, the infected larvae were prepared into sections and stained with ZIKV by immunohistochemistry assay. Viral particles were observed in the midgut, anal papilla, and other tissues. It suggested that ZIKV could infect the larvae of Ar. subalbatus via midgut and anal papilla.
The Ar. subalbatus adults were fed with dengue virus serotype 2 (DENV-2) containing blood and the larvae were reared in DENV-2-containing water in the same way. The results showed that no DENV-2 could be detected in vivo. Finally, the authors conclude that a strain of Ar. subalbatus isolated from Guangdong, China, can infect and transmit ZIKV but not DENV-2 under laboratory conditions. Moreover, the larvae of Ar. subalbatus can also be infected by ZIKV-contaminated urine. Exploring the vector competence of Ar. subalbatus for ZIKV and DENV provides an experimental basis for vector control and the prevention and control of mosquito-borne viral diseases.
