Molecular typing of Bluetongue Viruses using nCounter Analysis System platform Alfreda Tonelli , Stefano Gottardi, Cesare Cammà, Alessio Lorusso, Giovanni Savini
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Molecular typing of Bluetongue Viruses usingnCounter Analysis System platform
Alfreda Tonelli, Stefano Gottardi, Cesare Cammà, Alessio Lorusso, Giovanni Savini
Bluetongue Virus (BTV)
- BTV, Orbivirus/Reoviridae - 27 recognized serotypes- VP2 determines serotype specificity- Ten double-stranded RNA genetic
segments
Type specific RT-PCR
Serogroup identification (1-27)
ISOLATION SEROTYPING+
+KC, BHK, VERO
or
Diagnosis
BTV-4 BTV-16
BTV-9
BTV-1E
BTV-4
BTV-2
&BTV-4
BTV-1W BTV-15 1998
2001
19991978
1999
2006
2003
2002
2004
20062000
2002
2006
BTV incursions in the Mediterranean BTV incursions in the Mediterranean Basin from 1998 to 2006Basin from 1998 to 2006
LAB TESTS FOR BTV TEND TO BE PERFORMED BASED ON THE EPIDEMIOLOGICAL
SCENARIO
THEREFORE SOME VIRUS MAY BE MISSED !!
Microarray Imaging
Microarray Hybridization
Microarray Raw Data Analysis Microarray Statistical Data Analysis
BiologicalSamples
384-well plate
Microarray Production
Traditional Microarray
GOOGLE CAR
nCounter® Analysis System
nCounter® Analysis System
• Design of probes consists of 100 mer sequences- 58 capture probes
corresponding to the various Seg-2 sequences serotypes
- 2 capture probes one for NS1 and other for NS3 are included as positive controls
- 5 capture probes for Culicoides species
- 1 capture probe for actin gene
• The hybridization occurs in solution
• The probe pair consists of a - Reporter Probe, which carries
the signal on its 5’ end- Capture Probe which carries a
biotin on the 3’ end
nCounter® Analysis System
nCounter® Analysis System
RESULTS• The system enabled us to identify
- 25 (no BTV-25 and 27) reference isolates - BTV strains in tissue samples- BTV strains from pools of midges- Co-infections (BTV-1, BTV-4)
Experiments are still running to assess specificity and sensitivity
Future Prospects
nCounter® Analysis System may be used for DIAGNOSTICS- Identification of pathogen and co-infections- miRNA screening of in a pre-infection
process - Expression of key virulence genes in
pathogen and host (in an infection process)- Diagnostic chip for animal species
VIRUS DISCOVERY
MOLECULAR SURVEILLANCE IS CRUCIAL TO KEEP THE TEST UP-TO-DATE
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