Measurement of the infection and dissemination of bluetongue virus in culicoides biting midges using a semi-quantitative rt-PCR assay and isolation of infectious virus.
Measurement of the infection and dissemination of bluetongue virus in culicoides biting midges using a semi-quantitative rt-PCR assay and isolation of infectious virus.
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DOI：
10.1371/journal.pone.0070800
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Carpenter S
中科院分区：
文献类型：
--
作者：
Veronesi E;Antony F;Gubbins S;Golding N;Blackwell A;Mertens PP;Brownlie J;Darpel KE;Mellor PS;Carpenter S
库蠓（Culicoides biting midges）（双翅目：蠓科）是全球重要的家畜虫媒病毒的生物载体，包括蓝舌病毒（BTV）、非洲马瘟病毒（AHSV）和新近出现的施马伦贝格病毒（Schmallenberg virus，SBV）。从2006年至2009年，BTV在北方欧洲的爆发给农民造成了重大的破坏和经济损失，并进行了几次尝试以将古北界库蠓物种作为媒介。这些研究的结果很难解释，因为它们使用半定量RT-PCR（sqPCR）检测作为主要诊断工具，这种技术尚未经过验证可用于此目的。在本研究中，我们通过对库蠓属的两种菌落物种中的BTV RNA进行时间序列检测来验证这些检测方法的使用，并将结果与更传统的细胞培养物上的传染性BTV分离进行比较。将BTV血清1型毒株与马血混合，分别用不同浓度的马血喂养了数百只索诺库蠓和索诺库蠓。nubeculosus（Mg.）使用基于膜的测定，然后将充满的个体在25°C下孵育。每天从孵育中取出25只每种库蠓，均质化，并使用sqPCR（Cq值）和KC-C上的病毒分离定量每只库蠓中的BTV。sonorensis胚胎细胞系，然后进行抗原酶联免疫吸附测定（ELISA）。此外，还对全C. sonorensis和单独解剖的个人，以确定BTV传播的水平。从时间序列感染实验中产生的Cq值在两个C。sonorensis和C. nubeculosus证实了以前的研究，依赖于传染性BTV的分离和检测。现场收集的库蠓作为潜在的病毒载体的PCR检测和使用这种检测作为一线工具，用于在诊断实验室中的作用进行了讨论。
Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture. A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (Cq values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination. Cq values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.
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影响因子：
11.8
作者：
Elbers AR;Meiswinkel R;van Weezep E;Sloet van Oldruitenborgh-Oosterbaan MM;Kooi EA
通讯作者：
Kooi EA
影响因子：
2.2
作者：
Carpenter, S.;McArthur, C.;Mellor, P. S.
通讯作者：
Mellor, P. S.
影响因子：
2.1
作者：
Becker, M. E.;Reeves, W. K.;Foil, L. D.
通讯作者：
Foil, L. D.
影响因子：
2.7
作者：
Fu, H;Leake, CJ;Mellor, PS
通讯作者：
Mellor, PS
影响因子：
2.6
作者：
Ferrari, G;De Liberato, C;Autorino, GL
通讯作者：
Autorino, GL