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