References1. Allison DB, Cui X, Page GP, Sabripour M. 2006. Microarray data analysis: from disarray to consolidation and consensus. Nat Rev Genet; 7(1):55-65.2. Stricker TP, Morales La Madrid A, Chlenski A, Guerrero L, Salwen HR, Gosieng�ao Y, et al. 2014. Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounter system. Mol Oncol; 8(3):669-678.3. Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, et al. 2008. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol; 26(3):317-325.4. Quek SI, Ho ME, Loprieno MA, Ellis WJ, Elliott N, Liu AY. 2012. A multiplex assay to measure RNA transcripts of prostate cancer in urine. PLoS One; 7(9):e45656.5. Nanostring Technologies. nCounter® Product Literature. Overview of the nCounter Analysis System and nCounter Products. Nanostring Technologies (Rev. 4.0).6. Liew M, Seipp M, Ebbert M, Wilson A, Fauron C, Bastien R, et al. 2009. Poster: Performance of the nanostring nCounter compared to a quantitative Reverse Transcriptase Polymerase Chain Reaction Assay.7. Beechem JM. 2013. Poster: Single Cell Digital Gene Expression of 800 mRNA Targets Applications for Circulating Tumor Cells.8. Primary Industries Ministerial Council, Canberra, ACT. Animal Health Australia 2008. Disease strategy: Bluetongue (Version 3.0). Australian Veterinary Emergency Plan (AUSVETPLAN); 1:1-58.

Alfreda Tonelli1, Stefano Gottardi2, Cesare Cammà1, Alessio Lorusso1, Giovanni Savini1

1 OIE Reference Laboratory for Bluetongue, Istituto Zoopro�lattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy2 Diatech Pharmacogenetics srl, Jesi-Ancona, Italy

DiscussionSpeci�city of the assay needs to be further investigated against a larger panel of BTV samples collected worldwide and further experiments are currently ongoing to increase the sensitivity and speci�city of the test with the �nality to use the test in routine diagnostic laboratories.The nCounter® System has many advantages over other the major microarray platforms. The processing of samples of DNA or RNA does not require neither ampli�cation nor cloning therefore no gene-speci�c or 3’ biases are introduced thus reducing the risk of additional technical errors (3). The nCounter system has both the probe and target in solution rather than bound to a surface, this allows for a higher level of sensitivity across many target genes and lower amounts of starting material than in microarrays provides a digital readout of the amount of transcript in a sample (3). These counts are linear across a large dynamic range, they exhibit less background noise, and analysis of data is simple requiring only a normalization strategy. The analysis on the nCounter® System is less time-consuming than currently used tests (Figure 4). Analysis requires less e�ort and the nCounter® System is more scalable than real-time PCR or microarrays (3). The nCounter® System is less expensive and reproducible and it is versatile as other sequences of other viruses or sequences can be easily incorporated or added to the analysis making the test a multi-use diagnostic system (4). More importantly, the software of analysis is user-friendly, digital data output from this test can be easily archived and tracked, and, easily understood by the end-user.

IntroductionBluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of the family Reoviridae. BTV is transmitted mainly by Culicoides spp. midges. Twenty-six serotypes of BTV are described and a further serotype, the 27th, was recently reported in Corsica. Genome constellation of BTV is composed by 10 segments of dsRNA and among them, Seg-2 encodes for the VP2. VP2 is the outermost protein of the virion (Figure 1) and determines serotype speci�city eliciting in the host the production of serotype-speci�c protective antibodies. As for �u viruses, multiple BTV serotypes can co-circulate in the same region, as seen in Italy with BTV-1 and BTV-4 during 2014. Identi�cation of BTV serotype is performed by using multiple typing assays that tend to be executed based on the known epidemiological situation within a given country. Samples containing multiple serotypes (particularly those involving novel serotype introductions) may therefore be missed. The complete identi�cation of a single BTV strain from a sample can therefore be expensive and time consuming.

Aim of studyThe aim of this work is to develop a diagnostic test, based on the nCounter® Analysis System platform (Nanostring technologies), that would, with minimum hands on time and operator steps, simultaneously identify all BTV serotypes. The nCounter® Analysis System uses the principle of molecular hybridization, in combination with capture probes having a sequence-speci�c color code (that works as a molecular barcode as described in Figure 2). The nCounter® Analysis System should reveal infection and co-infections of BTV viruses in biological samples, including internal organs, blood specimens and in tissue culture adapted BTV strains.

The nCounter® Analysis System is a new microarray platform that utilizes color-coded molecular barcodes that digitally count nucleic acid molecules in solution. Microarray data is used for a wide variety of analyses as reviewed in Allison et al. 2006. These uses may be identi�cation of pathogens unsupervised clustering, classi�cation, di�erential expression analysis and expression of quantitative trait loci mapping (1). The nCounter® System has multiplex capability, and a user friendly digital readout (2).Materials and Methods

Probes (Figure 2) were designed according to the Seg-2 sequences available on line in NCBI. Reference BTV strains belonging to all serotypes but BTV-25 and BTV-27 and organs and blood from �eld samples with BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16 Italian strains were employed for the study. Figure 3 describes the entire work�ow process of the newly developed assay and the dynamics of hybridization.

ResultsExperiments were performed with the 25 reference BTV strains and with biological samples from the �eld. Biological samples were infected with BTV-1, BTV-2, BTV-4, BTV-9, and BTV-16. All samples were previously characterized by a serotype-speci�c real time RT-PCR. All viruses from �eld samples were correctly identi�ed as well as co-infections.

Molecular typing of Bluetongue Viruses usingnCounter Analysis System platform

9th Annual Meeting of EPIZONESeptember 2nd - 3rd 2015Le Corum Montpellier France

VP2

Figure 1

BarcodeHalf Site50 bases

Half Site50 bases

Figure 2

Hybridize1. Bu�er2. CodeSet3. Sample

Sample material is mixed with excessCodeSet and hybridized overnight.

Sample cartridges areplaced in the Digital Analyzerfor data collection. Fluorescentbarcodes on the surface of thecartridge are counted and a running total of each target is tabulated. Approximately 1 to 2 millionreads are generated in each lane of the �ow cell (12 lanes per cell).

The Prep Station is an automatedrobotic instrument that removes excessCodeSet and immobilizes CodeSet/RNA complexes in the nCounter Cartridge for data collection.

Purify

Count

1

2

3

Figure 3

Hybridization

Only 15 Minutesof Total

Hands-on Time

Hands-on Time 5 minutes

Day 1

5 minutes

Day 2 (automated)

5 minutes

Day 2 (automated)

Process

SampleProcessing

Digital DataAcquisition

Set Up Hybridization Set Up Prep Station Set Up Digital Analyzer

Add bu�er, CodeSet andsample into a strip tubeand hybridize overnight.

Place the strip tube onto theautomated nCounter PrepStation with reagents andconsumables from thenCounter Master Kit.

Take the cartridge from thePrep Station and place it intothe Digital Analyzer for directdigital counting.

1 2 3

Figure 4