Detection of Toggenburg Orbivirus by a segment 2-specific quantitative RT-PCR

Hofmann, Martin; Renzullo, Sandra; Planzer, Jeannette; Mader, Markus; Chaignat, Valérie; Thuer, Barbara

doi:10.1016/j.jviromet.2010.02.027

Cited by 20 publications

(12 citation statements)

References 11 publications

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“…In a second step, this mixture was used as matrix for an automated RNA extraction using NucleoSpin® 96 RNA kit. The RNA was then tested by real-time RT-PCR employing the S10-specific protocol able to detect all BTV-serotypes, inclusively TOV [48]. In S10-specific real-time RT-PCR positive samples, a serotype-8 specific commercial real-time RT-PCR was performed (TaqVet® Blue Tongue Virus Triplex – All Genotypes and BTV8, LSI, Lissieu, France) and if negative a TOV specific real-time RT-PCR was carried out [46].…”

Section: Methodsmentioning

confidence: 99%

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland

Casaubon

Chaignat²,

Vogt

et al. 2013

BMC Vet Res

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BackgroundIn 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests.ResultsTen out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening.ConclusionsPrevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.

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Section: Methodsmentioning

confidence: 99%

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland

Casaubon

Chaignat²,

Vogt

et al. 2013

BMC Vet Res

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“…the potential for cross-contamination. BTV 25 from Switzerland [4] and BTV 26 from By real-time RT-PCR the highly conserved Kuwait [5] have been, reported recently. In India 21 genome segment of ns1 can be targeted to detect all the different BTV serotypes have been reported from 24 BTV serotypes, as well as geographic variants different parts based upon serum neutralization and within the individual serotype.…”

Section: Introductionmentioning

confidence: 99%

Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

Vishwaradhya¹,

Minakshi²,

Ranjan³

et al. 2013

Vet World

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Aim: The study was conducted to develop ns1 gene based sensitive real-time reverse transcriptase PCR (real-time RT-PCR) assay for diagnosis of India isolates of bluetongue virus (BTV). Materials and Methods:The BTV serotype 21 isolate (KMNO7) was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA) of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the 10fold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results:The ns1 gene based group specific PCR showed a single 366 bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using 10 fold diluted viral RNA showed the detection limit of 70.0 fg (equivalent to 3 3.3x10 target copies of ns1 gene) per reaction in 1% agarose gel electrophoresis. The ns1 gene based real time RT-PCR was 2 successfully standardized and the detection limit was found to be 7.0 fg (equivalent to 3.3x10 target copies of ns1 gene) per reaction. Conclusion:The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR.

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“…There are 26 serotypes based on the outer protein VP2. These serotypes do not exhibit cross-protection (Hofmann et al 2010;Maan et al 2011a, b). Seven serotypes (BTV-1, BTV-2, BTV-3, BTV-4, have been identified in China.…”

Section: Introductionmentioning

confidence: 98%

DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1

Yang

et al. 2015

Appl Microbiol Biotechnol

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Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

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Detection of Toggenburg Orbivirus by a segment 2-specific quantitative RT-PCR

Cited by 20 publications

References 11 publications

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland

Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1

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