Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 17
APPLICATION OF REVERSE TRANSCRIPTION REAL-TIME PCR TO DETECT THE
SCHMALLENBERG VIRUS GENOME
Nikolai Salnikov, Helena Nikitina, Sodnom Tsybanov, Denis Kolbasov
National research institute for veterinary virology and microbiology of Russia, Pokrov, Russia
Schmallenberg disease, virus, PCR
Introduction
The disease Schmallenberg was registered among cattle, sheep
and goats in Germany, the Netherlands, Belgium, Britain, France,
Italy, Luxembourg and Spain (3,4). The Schmallenberg virus is
a member of the genus Orthobunyavirus family Bunyaviridae.
Established the pathogenicity of the Schmallenberg virus for
sheep, goats and cattle. Clinical signs of the disease in
cattle include fever, drop in milk yield (50%), loss of appetite and
sometimes diarrhea. If an adult ewe or heifer was infected in the
early stages of pregnancy, the fetal infection can occur,
leading to serious consequences: abortion, birth of premature or
dead fetuses and lambs, goats and calves with
various malformations. At detection in animals the characteristic
clinical signs of the Schmallenberg disease carry out
activities for virus isolation in cell cultures, detection of the
virus genome by PCR and antibodies to it in the neutralization
test (1,2). The aim of this work was the development the test -
system to detect the Schmallenberg virus genome by real-time
quantitative reverse transcription PCR (RT-qPCR).
Materials & methods
In our work the referent RNA samples of the Schmallenberg virus
were used. Also we used the different strains of such viruses, as
Nairobi sheep disease, Akabane, Rift Valley fever and
bluetongue. Extraction of viral RNA was performed using "Trizol''
(Trizol Reagent, Life Technology, USA). RT-qPCR was carried
out on the thermocycler Rotor Gene 6000 (Corbett Research,
Australia). Cloning of the amplified Schmallenberg virus
genome's fragment was performed using the "Ins TAclone PCR
cloning kit"(Fermentas, Latvia). The synthesis of RNA from
cloned DNA inserts was performed using the kit "RiboMAX Large
Scale RNA Production System T7" (Promega, USA).
preparations of heterologous viruses and RNA extracted from the
intact samples has not been received positive results. This
proves the specificity of the primers and the probe included in
the test - system. The analytical sensitivity of the developed test -
system is 7,178*10^5 RNA copies / mkl.
Acknowledgements
We thank for the referent RNA samples of the Schmallenberg
virus Dr. Martin Beer and Dr. Bernd Hoffman, Institute of
Diagnostic Virology, Friedrich Loeffler Institute, Germany.
References
1. Gibbens, N. Schmallenberg virus: a novel viral disease in northern
Europe. 2012. Vet. Rec.17: 58.
2. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier
H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg. Infect.
Dis. 2012 Mar.
3. Latest posts on Pro Med-mail. International society for infectious
diseases, 2011-2012. http://www.promedmail.org.
4. Weekly disease information/WAHID Interface/OIE, 2011-2012.-
http://www.oie.int.
Results
With order to select primers the available in Gen Bank nucleotide
sequences of Schmallenberg virus Genome were analyzed using
the programs Bio Edit 7.0 and Oligo 6.0. As a result a pair of the
primers flanking the fragment of 130 bp within the gene N of
Schmallenberg virus was designed. For detection of amplification
products in real-time the probe Taq-man, which contains at the
5'-end the fluorophores HEX and at the 3'-end – quencher BHQ2
was designed. Analytical sensitivity of the method was
determined using in vitro transcripts synthesized on the template
of the recombinant plasmid with the Schmallenberg virus
genome fragment's insertion. Ten-fold serial dilutions of in
vitro transcribed RNA (of known concentration) were used to
determine analytical sensitivity of RT-q PCR. The limit of
sensitivity considered the maximum dilution at which a positive
result was registered.
The calculated value of analytical sensitivity of RT-PCR in realtime
was 7.178*10^5 RNA copies /mkl, which corresponds
to 3,698*10^6 copies of RNA per reaction. To evaluate
the specificity of the test - system the RNA samples of Nairobi
sheep disease, Akabane, Rift Valley fever and bluetongue
viruses were examined. We also examined the RNA samples
extracted from the intact cultures of kidney cells of antelope,
BHK-21/13, CV-1, blood samples and organs from intact animals
(goats, sheep, cattle, mice). With any of the examined RNA
samples has not been a received positive result in RT-qPCR. By
using the developed method over 500 blood samples and 15
samples of placenta and meconium from clinically
healthy pregnant heifers imported from Germany and the
Netherlands in 2011-2012 in the farms of Nizhny Novgorod,
Moscow and Kaluga regions were examined. This also was
not obtained positive results.
Discussion & conclusions
The designed test -system is suitable to detect the
Schmallenberg virus genome in blood samples and
organs of sheep and cattle. In the investigation of RNA