Bluetongue Virus Detection by Real-Time RT-PCR inCulicoidesCaptured During the 2006 Epizootic in Belgium and Development of an Internal Control

Abstract

After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

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

“…The first, carried out in Belgium and utilizing small pools of pigmented Culicoides (<10 individuals/pool) recorded a C q range of 35.8–42.8 when utilising a cut-off value of 45 [19]. From the current study it appears likely that these ‘positive’ pools either contained no Culicoides with fully disseminated infections or that BTV RNA levels were reduced substantially during processing.…”

“…‘Positive’ findings of detected viral RNA may merely have represented inactivated BTV that had persisted in individual Culicoides following an infected blood meal, or a sub-transmissible infection such as commonly occurs in this genus [4]. Subsequent to these early studies, the inclusion of cycle threshold (C q ) values as a semi-quantitative indication of RNA concentration has become more common [18], [19]. Uncertainty remains, however, in interpretation of semi-quantitative or sqPCR data from pools of Culicoides and in the relationship between C q values representing quantity of viral RNA and the quantity of infectious virus as a means of defining transmissible infections.…”

Description of Culicoides (Culicoides) bysta n. sp., a new member of the Pulicaris group (Diptera: Ceratopogonidae) from Slovakia

“…chiopterus (Meigen, 1830); C. ( A. ) imicola Kieffer, 1913; C. ( Culicoides ) pulicaris (L.); and C. ( C. ) lupicaris Downes & Kettle, 1952 [2–10]. Within the subgenus Culicoides , C. ( C .)…”

Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR

“…A major challenge in interpreting arbovirus cycle threshold (C q ) values derived by sqPCR from pools of Culicoides lies in the fact that sub-transmissible infections are common in Culicoides occurring most frequently during initial infection and release from the hind mid-gut [15], [16]. This renders the numerous studies where only small numbers of positive pools of Culicoides are reported difficult to interpret [17], [18], [19], [20], [21], [22], although comparison with predicted quantities of virus in the original blood meal in larger studies can be used to demonstrate that at least limited replication of virus has occurred in the putative vector and that wide-scale infection of Culicoides did occur [23].…”