Development of One step Multiplex RT-PCR for differentiation of FMDV serotypes A, O and SAT2 in Egypt Amir A. Shehata.

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Development of One step Multiplex RT-PCR for differentiation of FMDV serotypes A, O and SAT2 in Egypt Amir A. Shehata

FMD distribution pattern

FMD situation in Egypt O/ME-SA/Egy-72 A/Africa/G-VII (2006 & 2009) O/ME-SA/PanAsia 2 A/Asia/Iran-05 Bar-08 (2010) SAT2/VII/Ghb-12 (2012) SAT2/VII/Alx-12 (2012) O/EA-3 (2012) A/Africa/G-IV (ISM-12) (2012) Donald King (FMD Global and Regional update, 2014)

FMDV RT-PCR target conserved IRES and 3D regions variable VP1 region * putative functions Membrane-binding Genome-linked (VPg) 5’UTR Carboxy-terminal self-cleaving 3’UTR Protease Capsid NTP binding* Protease Polymerase VPG L 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D VP4 VP2 VP3 VP1 AAA (n) Poly(C) Primary cleavages L 2A 3C Secondary cleavages 1B/RNA? 3C 3C 3C 3C 3C 3C Brief update of recent improvements in FMDV diagnosis Move on to discuss the development of pen-side assays for the detection of FMDV conserved IRES and 3D regions targets for pan-serotype reactive assays 1 variable VP1 region -sequences used for isolate characterisation 2 3 4 5 6 7 8 Kilobases 4

One Step Multiplex RT-PCR Strategy VP1 2A 2B A 750 bp SAT2 666 bp O 283 bp

2- Pan-serotypic detection of FMDV RNA (Reid et al.,2000) Methods 1- RNA Extraction 2- Pan-serotypic detection of FMDV RNA (Reid et al.,2000) 3- Assay Validation

Assay Validation To check specificity and / or cross reactivity: 7 archival samples representing the strains recorded in Egypt except A/ Africa/G-VII. BHK-21 cell culture and Epithelial tissue from FMD negative cattle and buffalo. 45 clinical and T/C samples collected from 2012 till 2016 were tested by Ag ELISA and the developed assay. - Confirmation by nucleotide sequencing of amplified products. Determination of Limit of Detection (LOD)

Determination Of Specificity O/ME-SA/Pan-Asia 2 O/ME-SA/Sharqia-72 SAT2/VII/Ghb-12 SAT2/VII/Alx-12 A/Asia/Iran-05 A/Africa/G-IV Mixed sample C -Ve Marker O/ EA-3 750 bp 666 bp 500 bp 200 bp 283 bp

A SAT2 O NTC M A SAT2 O

S SAT2 EA-3 2015 2014 A O mixed A&O O/EA-3 O O panasia2 A /Iran-05 M O Sharqia-72 M O/EA-3 2014 NTC O EA-3 2015 SAT2 750 bp 500 bp A 666 bp 283 bp 200 bp 25-2-2015 mpcr for seminar O

M SAT2/ Alx-12 NTC G-IV O/EA-3 (2016) A /Iran-05 A/ Africa/ 500 bp

S BHK-21 Buffalo Cattle A +ve control 500 bp 200 bp

Limit Of Detection Serotype O M 106 105 104 103 102 10 NTC 500 bp

Serotype SAT2 M 106 105 104 103 102 10 NTC 666 bp 500 bp

Serotype A M 106 105 104 103 102 10 NTC 750 bp 500 bp

Conclusion & Recommendation The developed Multiplex RT-PCR assay differentiate accurately between FMDV A, O and SAT2 serotypes in Egypt and will save cost, time and effort. It is highly recommended to continuously monitor the circulating strains by nucleotide sequencing to be sure that the developed assay is fit for purpose and to detect any further changes in the circulating strains.

Kees van Maanen (EuFMD) Acknowledgement Special Thanks Kees van Maanen (EuFMD) Animal Health Research Institute: Hanaa A. Ahmed Faculty of veterinary medicine – Cairo University : Mohamed A. Shalaby. Ayman H. El-Deeb. Swedish University of Agricultural Sciences : - Jean-Francois Valarcher