Arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing.
Arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing.
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DOI：
10.1371/journal.pntd.0000878
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发表时间：
2010-11-09
影响因子：
3.8
通讯作者：
Read TD
中科院分区：
文献类型：
--
作者：
Bishop-Lilly KA;Turell MJ;Willner KM;Butani A;Nolan NM;Lentz SM;Akmal A;Mateczun A;Brahmbhatt TN;Sozhamannan S;Whitehouse CA;Read TD
尽管节肢动物传播的病毒对全球造成了威胁，但目前还没有一种有效的方法来筛选媒介种群来检测新的病毒序列。目前基于培养的病毒检测和监测方法可能既昂贵又耗时，而且是基于对病原体的先验知识，因为它们依赖于特定的寡核苷酸引物或抗体。因此，这些技术可能不适合于暴发的病原体未知的情况。在这项研究中，我们探索了使用高通量焦磷酸测序来监测节肢动物传播的RNA病毒。登革病毒是正链RNA黄病毒家族的成员，由伊蚊属的几个成员传播，作为模型使用。实验感染了1型登革病毒(DENV-1)的埃及伊蚊与未感染的蚊子共用一组，以模拟从正在进行的虫媒病毒监测项目中提取的样本。采用随机扩增的方法，对总RNA进行逆转录，得到的cDNA经454焦磷酸测序。在两种类型的样本中，一种是5只感染DENV-1的成年蚊子，另一种是1只感染DENV-1的蚊子和4只未感染的蚊子，我们鉴定了DENV-1的DNA序列。在一个由5只成年蚊子组成的未受感染的控制池中，没有检测到DENV-1序列。我们计算了Ae的比例。每个感染登革病毒基因组的埃及伊蚊后基因组的贡献率在2.75×10−8~1.0 8×10−7之间。登革热病毒1RNA在蚊子体内高度集中，利用现有的高通量测序仪器是可行的。我们还根据表达的RNA序列鉴定了蚊子微生物区系的一些成分。这包括细菌属Pirellula和Asaia的成员，各种真菌，以及一种潜在的未确定特征的真菌病毒。传统的病毒检测方法通常依赖于病毒的特定属性，例如DNA序列，因此它们不仅需要所讨论的病原体的先验知识，而且它们通常也是非常特定的，例如仅能够从特定家族中检测病毒。NextGen测序由于其不断增加的吞吐量、降低的成本和不偏不倚的性质，在检测/诊断应用方面显示出很大的前景。我们以实验接种登革病毒1型(DENV-1)的埃及伊蚊为对象，研究了454焦磷酸测序在昆虫种群病毒监测中的适用性，并计算了病毒在被粉碎的蚊子总核酸中所占的比例。我们的结论是，454焦磷酸测序能够从感染和未感染的蚊子池中检测到即使是非常少量的已知病毒，但对于检测病毒所需的测序读数来说，这项技术目前可能成本太高，不能用于大规模的监测工作。我们研究的有趣副产品包括了解共生生物是什么。埃及伊蚊可能含有，以及在感染了DENV-1的蚊子和未感染的蚊子中可能存在哪些基因的差异表达。
Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown. In this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing. In two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (pIP), which ranged from 2.75×10−8 to 1.08×10−7. DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus. Traditional methods for virus detection often rely on specific attributes, such as DNA sequences, of the viruses and therefore they not only require a priori knowledge of the agent in question, but they also are generally very specific in nature, capable of detecting viruses only from within a specific family, for example. Nextgen sequencing shows much promise for detection/diagnostic applications because of its ever-increasing throughput, decreasing cost, and unbiased nature. We investigated the applicability of 454 pyrosequencing for viral surveillance of insect populations, using Aedes aegypti mosquitoes experimentally inoculated with Dengue virus type 1 (DENV-1) and calculated what proportion of the total nucleic acid from crushed mosquitoes was contributed by the virus. We concluded that 454 pyrosequencing is capable of detecting even very small amounts of a known virus from within a pool of infected and noninfected mosquitoes, but for the amount of sequencing reads required to detect the virus, this technique may currently be too cost-prohibitive for use in large-scale surveillance efforts. Interesting byproducts of our study included a glimpse into what symbiotic organisms Ae. aegypti may harbor, as well as what genes may be differentially expressed in a DENV-1-infected versus noninfected mosquito.
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DOI：
10.1073/pnas.0610451104
发表时间：
2007-05-22
影响因子：
11.1
作者：
Favia, Guido;Ricci, Irene;Daffonchio, Daniele
通讯作者：
Daffonchio, Daniele
影响因子：
4.4
作者：
Fuchsman, Clara A.;Rocap, Gabrielle
通讯作者：
Rocap, Gabrielle
影响因子：
3.2
作者：
LIESACK, W;STACKEBRANDT, E
通讯作者：
STACKEBRANDT, E
影响因子：
14.9
作者：
Cole JR;Wang Q;Cardenas E;Fish J;Chai B;Farris RJ;Kulam-Syed-Mohideen AS;McGarrell DM;Marsh T;Garrity GM;Tiedje JM
通讯作者：
Tiedje JM
DOI：
10.1073/pnas.1431443100
发表时间：
2003-07-08
影响因子：
11.1
作者：
Glöckner, FO;Kube, M;Reinhardt, R
通讯作者：
Reinhardt, R