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Culicoids as vectors of Schmallenberg virus.

Rasmussen LD, Kristensen B, Kirkeby C, Rasmussen TB, Belsham GJ, Bødker R, Bøtner A - Emerging Infect. Dis. (2012)

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Pools of culicoids were homogenized in water (100 µL) by using a 3-mm stainless steel bead (Dejay Distribution Ltd., Launceston, UK) in a TissueLyser II (QIAGEN, Hilden, Germany) for 1 min at 25 Hz... Two separate 1-step reverse transcription quantitative PCRs (RT-qPCRs), targeting the L segment and the S segment of SBV RNA, were performed according to protocols provided by the Friedrich Loeffler Institute in Germany on the extracted nucleic acids by using a Mx3005p qPCR system (Agilent Technologies, Palo Alto, CA, USA)... Another RT-qPCR targeting ruminant β-actin mRNA was performed as an internal endogenous control... This uptake of blood should therefore lead to a Ct value that is at least 6–7 units higher (≈100-fold lower level of RNA) when a single midge is tested by RT-qPCR... Thus, even if all 5 culicoids in a pool had recently taken a blood meal from a viremic animal, the Ct values observed here strongly suggest replication of SBV within the C. obsoletus group midges... However, in principle, other hosts of SBV could have a much higher level of viremia than cattle and could provide the levels of SBV RNA detected... C. punctatus s.s. midges cannot be ruled out as a possible vector of SBV because of the limited number of insects tested... Our study demonstrates the presence of SBV RNA in C. obsoletus group midges caught in Denmark during October 2011... The low Ct values (i.e., high SBV RNA levels) and the absence of ruminant β-actin mRNA in these samples strongly suggest that SBV replicates in these midges and hence that the C. obsoletus group midges are natural vectors for this virus.

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RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, C. obsoletus group midge pools 1–8; lanes 9–10; negative and positive controls, respectively. Numbers below lanes are cycle threshold values from RT-qPCRs; –, no value. M, size marker. Amplicons (145 bp) from positive pools were extracted and sequenced.
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Figure 1: RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, C. obsoletus group midge pools 1–8; lanes 9–10; negative and positive controls, respectively. Numbers below lanes are cycle threshold values from RT-qPCRs; –, no value. M, size marker. Amplicons (145 bp) from positive pools were extracted and sequenced.

Mentions: Two of 22 pools tested strongly positive for the large (L) and small (S) segments of SBV RNA. Each positive sample was derived from 5 midges of the C. obsoletus group. One pool produced cycle threshold (Ct) values of 26.4 and 24.5 (in the L segment– and S segment–specific assays, respectively), whereas the second positive pool gave Ct values of 28.8 (L segment) and 27.6 (S segment). These pools were negative for the internal endogenous control that targeted the bovine/ovine β-actin mRNA. This result makes it unlikely that the detection of SBV RNA within the midges resulted from recent blood meals from infected animals remaining within the culicoids and suggests the virus has replicated within the midges. The PCR amplicons (145 bp; Figure) from the L segment–specific RT-qPCR were sequenced by using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of 80 bp from the amplicons, excluding the primer sequences, had 100% identity with the expected region of the SBV segment L (1).


Culicoids as vectors of Schmallenberg virus.

Rasmussen LD, Kristensen B, Kirkeby C, Rasmussen TB, Belsham GJ, Bødker R, Bøtner A - Emerging Infect. Dis. (2012)

RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, C. obsoletus group midge pools 1–8; lanes 9–10; negative and positive controls, respectively. Numbers below lanes are cycle threshold values from RT-qPCRs; –, no value. M, size marker. Amplicons (145 bp) from positive pools were extracted and sequenced.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376822&req=5

Figure 1: RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, C. obsoletus group midge pools 1–8; lanes 9–10; negative and positive controls, respectively. Numbers below lanes are cycle threshold values from RT-qPCRs; –, no value. M, size marker. Amplicons (145 bp) from positive pools were extracted and sequenced.
Mentions: Two of 22 pools tested strongly positive for the large (L) and small (S) segments of SBV RNA. Each positive sample was derived from 5 midges of the C. obsoletus group. One pool produced cycle threshold (Ct) values of 26.4 and 24.5 (in the L segment– and S segment–specific assays, respectively), whereas the second positive pool gave Ct values of 28.8 (L segment) and 27.6 (S segment). These pools were negative for the internal endogenous control that targeted the bovine/ovine β-actin mRNA. This result makes it unlikely that the detection of SBV RNA within the midges resulted from recent blood meals from infected animals remaining within the culicoids and suggests the virus has replicated within the midges. The PCR amplicons (145 bp; Figure) from the L segment–specific RT-qPCR were sequenced by using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of 80 bp from the amplicons, excluding the primer sequences, had 100% identity with the expected region of the SBV segment L (1).

View Article: PubMed Central - PubMed

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Pools of culicoids were homogenized in water (100 µL) by using a 3-mm stainless steel bead (Dejay Distribution Ltd., Launceston, UK) in a TissueLyser II (QIAGEN, Hilden, Germany) for 1 min at 25 Hz... Two separate 1-step reverse transcription quantitative PCRs (RT-qPCRs), targeting the L segment and the S segment of SBV RNA, were performed according to protocols provided by the Friedrich Loeffler Institute in Germany on the extracted nucleic acids by using a Mx3005p qPCR system (Agilent Technologies, Palo Alto, CA, USA)... Another RT-qPCR targeting ruminant β-actin mRNA was performed as an internal endogenous control... This uptake of blood should therefore lead to a Ct value that is at least 6–7 units higher (≈100-fold lower level of RNA) when a single midge is tested by RT-qPCR... Thus, even if all 5 culicoids in a pool had recently taken a blood meal from a viremic animal, the Ct values observed here strongly suggest replication of SBV within the C. obsoletus group midges... However, in principle, other hosts of SBV could have a much higher level of viremia than cattle and could provide the levels of SBV RNA detected... C. punctatus s.s. midges cannot be ruled out as a possible vector of SBV because of the limited number of insects tested... Our study demonstrates the presence of SBV RNA in C. obsoletus group midges caught in Denmark during October 2011... The low Ct values (i.e., high SBV RNA levels) and the absence of ruminant β-actin mRNA in these samples strongly suggest that SBV replicates in these midges and hence that the C. obsoletus group midges are natural vectors for this virus.

Show MeSH
Related in: MedlinePlus