OS18 - Open Session 2018 Borgo Egnazia (Puglia), Italy 29 - 31 /10/2018 EuFMD Sessions\Open Session\Archive-2018\Open 2018 - Italy\6. Online & Streaming\PPTs

1. RAPID, ON SITE, DIAGNOSIS OF FMD AND SAFE AND
COST-EFFECTIVE SHIPMENT OF SAMPLES USING LATERAL
FLOW DEVICES FOR LABORATORY DIAGNOSTICS
Bakkali Kassimi L1, Belsham, G.J.2, Bulut A.N.3, Gorna, K.1, Hamers, C.4, Hudelet, P.4, Jamal, S.5, Laloy, E.1, Relmy, A.1,
Romey, A.1, Ularamu H.G.6, Zientara, S.1 and Blaise-Boisseau S.1
1Anses, France, 2DTU, Denmark, 3SAP/FMD Institute, Turkey, 4Boehringer Ingelheim, France,
5University of Malakand, Pakistan, 6 NVRI, Nigeria
Global Vaccine Security

2. Towards global control and eradication of FMD
https://www.researchgate.net/publication/305238243_Genomics_and_outbreaks_Foot_and_mouth_disease; April, 2016.
Global Vaccine Security - OS18: Oct 2018, Italy

3. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

4. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

5. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

6. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

7. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

8. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Use of lateral flow device for safe and low cost
shipment of FMDV suspected samples (FMDVINACT)

9. Towards global control and eradication of FMD
Global Vaccine Security - OS18: Oct 2018, Italy
Use of lateral flow device for safe and low cost
shipment of FMDV suspected samples (FMDVINACT)

10. Experimental protocol
Global Vaccine Security - OS18: Oct 2018, Italy
Viral
suspension
Elution
Inoculation on cells
30min
Soaking
15min
in 0.2%
C6H8O7

11. Experimental protocol
Global Vaccine Security - OS18: Oct 2018, Italy
Viral
suspension
RNA
extraction
Elution
Inoculation on cells
RT-rtPCR 3D & IRES
rtPCR VP1 & sequencing
30min
Soaking
15min
in 0.2%
C6H8O7
3DL VP4 VP2 VP3 2A 2B 2C 3A 3B 3CVP1
VPg5’ 3’
IRES
3B 3B
Structural proteins Non-structural proteins

12. Experimental protocol
Global Vaccine Security - OS18: Oct 2018, Italy
Viral
suspension
RNA
extraction
Elution
Inoculation on cells
RT-rtPCR 3D & IRES
rtPCR VP1 & sequencing
Chemical
transfection
Virus
rescue
30min
Soaking
15min
in 0.2%
C6H8O7
3DL VP4 VP2 VP3 2A 2B 2C 3A 3B 3CVP1
VPg5’ 3’
IRES
3B 3B
Structural proteins Non-structural proteins

13. Experimental protocol
Global Vaccine Security - OS18: Oct 2018, Italy
Viral
suspension
RNA
extraction
Elution
Inoculation on cells
RT-rtPCR 3D & IRES
rtPCR VP1 & sequencing
Chemical
transfection
Virus
rescue
30min
Soaking
15min
in 0.2%
C6H8O7
3DL VP4 VP2 VP3 2A 2B 2C 3A 3B 3CVP1
VPg5’ 3’
IRES
3B 3B
Structural proteins Non-structural proteins
• 7 reference strains
• 3 archival samples

14. Field evaluation
o Anses, France (coordinator)
o Technical University of Denmark (DTU)
o FMD Research Centre of Nigeria (NRVI)
o FMD Institute of Turkey (SAP)
o University of Malakand in Pakistan (UM)
o Merial- Boehringer Ingelheim (BI)
Evaluation in field conditions of a safe and cost-effective
protocol for shipment of samples from FMD suspected
cases for laboratory diagnostic (FIELD_EVAL_INACT)
Global Vaccine Security - OS18: Oct 2018, Italy

15. Discard
citric acid
In farm
Global Vaccine Security - OS18: Oct 2018, Italy
Field evaluation on freshly collected clinical samples

16. Decontamination
solution
S1 S2 S3
In decontamination area
Discard
citric acid
In farm
Global Vaccine Security - OS18: Oct 2018, Italy
Field evaluation on freshly collected clinical samples

17. Decontamination
solution
S1 S2 S3
In decontamination area
Discard
citric acid
Triple packaging
At room T
S1
S2
S3
In clean area
In farm
Global Vaccine Security - OS18: Oct 2018, Italy
Field evaluation on freshly collected clinical samples

18. Field evaluation on freshly collected clinical samples
Global Vaccine Security - OS18: Oct 2018, Italy

19. Samples collection and inactivation of LFD in the field
Global Vaccine Security - OS18: Oct 2018, Italy
Turkey, n=20, completed
Nigeria, n=20, completed
Pakistan, n=400, in progress

20. Samples collection and inactivation of LFD in the field
Global Vaccine Security - OS18: Oct 2018, Italy
Turkey, n=20, completed
Nigeria, n=20, completed
Pakistan, n=400, in progress
Ularamu Hussaini, Nigeria, 2018 Ularamu Hussaini, Nigeria, 2018 Ularamu Hussaini, Nigeria, 2018

21. Field evaluation on freshly collected clinical samples
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018 Ularamu Hussaini, Nigeria, 2018Ularamu Hussaini, Nigeria, 2018
Naci Bulut, Turkey, 2018 Ularamu Hussaini, Nigeria, 2018 Naci Bulut, Turkey, 2018

22. Safety tests in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Nigeria
Turkey

23. Safety tests in the lab
9Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Nigeria
Turkey
… in progress
Elution Inoculation
on cells
15min
in 0.2%
C6H8O7

24. Molecular detections in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Netherlands

25. Molecular detections in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Netherlands
… in progress
15min
in 0.2%
C6H8O7
RNA
extraction
RT-rtPCR
3D &
IRES
rtPCR VP1
&
sequencing

26. Virus rescue in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Netherlands

27. Virus rescue in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Netherlands
… in progress
RNA
extraction
15min
in 0.2%
C6H8O7
Chemical
transfection

28. Virus rescue in the lab
Global Vaccine Security - OS18: Oct 2018, Italy
Denmark
France
Netherlands
… in progress
RNA
extraction
15min
in 0.2%
C6H8O7
Chemical
transfection
Optimization step

29. Optimization of RNA chemical transfection
Global Vaccine Security - OS18: Oct 2018, Italy
Chemical
Reagents
RNA/ transfectants
ratios
CPE on cells 24 hpt
IBRS-2 ZZ-R-127 BHK-21
Lipo 2000
1 + ++ -
2 ++ +++ -
3 ++ +++ -
4 ++ +++ -
5 - + -
6 - ++ -
7 - ++ -
8 - ++ -
Lipo 3000
1 - + -
2 +/- ++ -
3 + ++ -
4 + ++ -
5 - +/- -
6 - + -
7 - +/- -
8 - +/- -
Messenger
MAX
1 +++ ++ -
2 +++ ++ -
3 +++ +++ -
4 Toxicity +++ -
5 - ++ -
6 - ++ -
7 - ++ -
8 - ++ -
Trans-IT
1 +++ ++ +/-
2 +++ ++ +/-
3 +++ + +/-
4 +++ + +/-
5 +/- +/- -
6 +/- +/- -
7 +/- +/- -
8 +/- +/- -
Transfast
1 ++ ++ +/-
2 ++ ++ +/-
3 ++ ++ +/-
4 +/- Toxicity -
5 +/- Toxicity -
6 +/- Toxicity -
Optimization step
- 5 transfection reagents
(Biswal et al., 2015; Bisht et al., 2014…)
- 8 conditions per reagent
- 3 cells lines

30. Optimization of RNA chemical transfection
Global Vaccine Security - OS18: Oct 2018, Italy
Chemical
Reagents
RNA/ transfectants
ratios
CPE on cells 24 hpt
IBRS-2 ZZ-R-127 BHK-21
Lipo 2000
1 + ++ -
2 ++ +++ -
3 ++ +++ -
4 ++ +++ -
5 - + -
6 - ++ -
7 - ++ -
8 - ++ -
Lipo 3000
1 - + -
2 +/- ++ -
3 + ++ -
4 + ++ -
5 - +/- -
6 - + -
7 - +/- -
8 - +/- -
Messenger
MAX
1 +++ ++ -
2 +++ ++ -
3 +++ +++ -
4 Toxicity +++ -
5 - ++ -
6 - ++ -
7 - ++ -
8 - ++ -
Trans-IT
1 +++ ++ +/-
2 +++ ++ +/-
3 +++ + +/-
4 +++ + +/-
5 +/- +/- -
6 +/- +/- -
7 +/- +/- -
8 +/- +/- -
Transfast
1 ++ ++ +/-
2 ++ ++ +/-
3 ++ ++ +/-
4 +/- Toxicity -
5 +/- Toxicity -
6 +/- Toxicity -
MockCPE on ZZ-R-127 48h post-transfection

31. Optimization of RNA chemical transfection
Global Vaccine Security - OS18: Oct 2018, Italy
Chemical
Reagents
RNA/ transfectants
ratios
CPE on cells 24 hpt
IBRS-2 ZZ-R-127 BHK-21
Lipo 2000
1 + ++ -
2 ++ +++ -
3 ++ +++ -
4 ++ +++ -
5 - + -
6 - ++ -
7 - ++ -
8 - ++ -
Lipo 3000
1 - + -
2 +/- ++ -
3 + ++ -
4 + ++ -
5 - +/- -
6 - + -
7 - +/- -
8 - +/- -
Messenger
MAX
1 +++ ++ -
2 +++ ++ -
3 +++ +++ -
4 Toxicity +++ -
5 - ++ -
6 - ++ -
7 - ++ -
8 - ++ -
Trans-IT
1 +++ ++ +/-
2 +++ ++ +/-
3 +++ + +/-
4 +++ + +/-
5 +/- +/- -
6 +/- +/- -
7 +/- +/- -
8 +/- +/- -
Transfast
1 ++ ++ +/-
2 ++ ++ +/-
3 ++ ++ +/-
4 +/- Toxicity -
5 +/- Toxicity -
6 +/- Toxicity -
MockCPE on ZZ-R-127 48h post-transfection
2 transfection reagents
1 condition per reagent
1 cell line
… in progress

32. Conclusion
Global Vaccine Security - OS18: Oct 2018, Italy
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

33. Conclusion
Global Vaccine Security - OS18: Oct 2018, Italy
Soaking
15min
in 0.2%
C6H8O7
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

34. Conclusion
Global Vaccine Security - OS18: Oct 2018, Italy
Soaking
15min
in 0.2%
C6H8O7
Romey,Guinée,2018
Ularamu Hussaini, Nigeria, 2018
Ularamu Hussaini, Nigeria, 2018

35. Conclusion
Global Vaccine Security - OS18: Oct 2018, Italy
Soaking
15min
in 0.2%
C6H8O7
Effective
vaccine
selection
UlaramuHussaini,Nigeria,2018
Romey,Guinée,2018
Ularamu Hussaini, Nigeria, 2018

36. Conclusion
Global Vaccine Security - OS18: Oct 2018, Italy
Soaking
15min
in 0.2%
C6H8O7
Effective
vaccine
selection
UlaramuHussaini,Nigeria,2018
Romey,Guinée,2018
Ularamu Hussaini, Nigeria, 2018

37. Grazie a tutti per l’attenzione e per l'organizzazione