1. MOLECULAR SEROTYPING OF BLUETONGUE VIRUS ISOLATES FROM THE 2014/2015 SEASON IN SOUTH AFRICA K. GOOSEN; F. VAN DER MEER; I. RAUTENBACH; A. BOTHA; D GOOSEN* *Corresponding Author http://www.stackyard.com/news/images/fao/bluetongue.jpg 1

2. PRIMER DESIGN 15 BTV REFERENCE SEROTYPES PCR OPTIMIZATION BOTTLE A SINGLE SEROTYPING PCR MULTIPLEX A BOTTLE A PCR PCR OPTIMIZATION BOTTLE B AND C BOTTLE B AND C SINGLE SEROTYPING PCR MULTIPLEX B MULTIPLEX C FINAL PCR CONDITIONS FOR BOTTLE A,B,C MULTIPLEX PCR’S CONFIRM PCR CONDITIONS CONFIRM WORKING PRIMER CONCENTRATIONS FLOW DIAGRAM 2

3. PRIMER DESIGN https://upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_Re vComp.svg/2000px-Primers_RevComp.svg.png 3

4. PRIMER DESIGN Based on 15 BTV reference serotypes found in Southern Africa OBP Vaccine (A,B & C) containing these 15 reference serotypes were used as template for molecular serotype development A - 1,4,6,12 and 14 B - 3,8,9,10 and 11 C – 2,5,7,13 and 19 Primers designed to amplify as follow: Serotype 1 – 100 to 200 bp Serotype 2 – 200 to 300 bp Serotype 3 – 300 to 400 bp Serotype 4 – 400 to 500 bp Serotype 5 – 500 to 600 bp ETC. 4

5. PRIMER DESIGN 15 BTV REFERENCE SEROTYPES PCR OPTIMIZATION BOTTLE A SINGLE SEROTYPING PCR MULTIPLEX BOTTLE A PCR FLOW DIAGRAM 5

6. BOTTLE A SINGLE SEROTYPING PCR ALL PRIMERS 0.4 µM 6 4 14 12 1 95°C 55°C 72°C 4°C 1 Min15 sec 10 sec1 Min 35 Cycles 6 500 BP 1000 BP

7. BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR DIFFERENTIAL PRIMER CONCENTRATIONS 1- 0.2 µM 4- 0.1 µM 6- 0.2 µM 12- 0.6 µM 14- 0.6 µM 95°C 55°C 72°C 4°C 1 Min15 sec 12 sec1 Min 35 Cycles 7

8. BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR MULTIPLEX PRIMER CONCENTRATIONS 1- 0.2 µM 4- 0.1 µM 6- 0.2 µM 12- 0.6 µM 14- 0.6 µM 95°C 55°C 72°C 4°C 1 Min15 sec 90sec1 Min 35 Cycles 6 4 14 12 1 8

9. PRIMER DESIGN 15 BTV REFERENCE SEROTYPES PCR OPTIMIZATION BOTTLE A SINGLE SEROTYPING PCR MULTIPLEX BOTTLE A PCR PCR OPTIMIZATION BOTTLE B AND C BOTTLE B AND C SINGLE SEROTYPING PCR MULTIPLEX B MULTIPLEX C FLOW DIAGRAM 9

10. BOTTLE B AND C SINGLE SEROTYPING PCR ALL PRIMERS 0.4 µM 95°C 55°C 72°C 4°C 1 Min15 sec 1 Min 35 Cycles 389 10 11 257 1319 10

11. BOTTLE B AND C SINGLE SEROTYPING PCR SEROTYPES 7, 8, 9, 10, 11, 13 & 19 ALL PRIMERS 0.6 µM 89 10 11 7 1319 7 11

12. BOTTLE B AND C MULTIPLEX SEROTYPING PCR all primers 0.6 µM 89 10 11 7 13 7 19CB 12

13. PRIMER DESIGN 15 BTV REFERENCE SEROTYPES PCR OPTIMIZATION BOTTLE A SINGLE SEROTYPING PCR MULTIPLEX BOTTLE A PCR PCR OPTIMIZATION BOTTLE B AND C BOTTLE B AND C SINGLE SEROTYPING PCR MULTIPLEX B MULTIPLEX C FINAL PCR CONDITIONS FOR BOTTLE A,B,C MULTIPLEX PCR’S CONFIRM PCR CONDITIONS CONFIRM WORKING PRIMER CONCENTRATIONS FLOW DIAGRAM 13

14. FINAL MULTIPLEX SEROTYPING PCR’S 95°C 55°C 72°C 4°C 1 Min15 sec 90sec1 Min 35 Cycles MULTIPLEX PRIMER CONCENTRATIONS BOTTLE A 1- 0.2 µM 4- 0.1 µM 6- 0.2 µM 12- 0.6 µM 14- 0.6 µm MULTIPLEX PRIMER CONCENTRATIONS BOTTLE B 3- 0.1 µM 8- 0.5 µM 9- 0.5 µM 10- 0.6 µM 11- 0.6 µM MULTIPLEX PRIMER CONCENTRATIONS BOTTLE C 2- 0.1 µM 5- 0.1 µM 7- 0.5 µM 13- 0.6 µM 19- 0.6 µM ABC 14

15. PRIMER DESIGN 15 BTV REFERENCE SEROTYPES PCR OPTIMIZATION BOTTLE A SINGLE SEROTYPING PCR MULTIPLEX BOTTLE A PCR PCR OPTIMIZATION BOTTLE B AND C BOTTLE B AND C SINGLE SEROTYPING PCR MULTIPLEX B MULTIPLEX C FINAL PCR CONDITIONS FOR BOTTLE A,B,C MULTIPLEX PCR’S CONFIRM PCR CONDITIONS CONFIRM WORKING PRIMER CONCENTRATIONS BTV SEROTYPING OF DCA FIELD ISOLATES 2014/2015 WITH THE OPTIMIZED PCR FIRST A, B & C MULTIPLEX PCR CONFIRM SUSPECTED HITS WITH SINGLE SEROTYPING PCR FLOW DIAGRAM 15

16. DCA # 26529 MULTIPLEX A, B & C ABC 26529 SINGLE SEROTYPING 1, 3, 4, 6, 13 & 19 1 3 46 13 16 1,4,6 3 13/19 19 26529

17. DCA # 26529 MULTIPLEX HITS 1 3 4 6 13/19* * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 1 17 RESULT: DCA # 26529 CONTAINS BTV SEROTYPE 1

18. DCA # 26650 MULTIPLEX A, B & C ABC 26650 SINGLE SEROTYPING 1, 3, 13 & 19 1313 18 1 3 19 13/19 26650

19. DCA # 26650 MULTIPLEX HITS 1 3* 13/19* * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 1 19 RESULT: DCA # 26650 CONTAINS BTV SEROTYPE 1

20. DCA # 26728 MULTIPLEX A, B & C ABC 26728 SINGLE SEROTYPING 4 & 6 46 20 4/6 26728

21. DCA # 26728 MULTIPLEX HITS 4/6 * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 6 21 RESULT: DCA # 26728 CONTAINS BTV SEROTYPE 6

22. SINGLE SEROTYPING 3, 5, 7, 13 & 19 35 26797 71319 DCA # 26797 MULTIPLEX A, B & C ABC 26797 22 4/6 13/19 5,7 3

23. DCA # 26797 MULTIPLEX HITS 3* 5* 7* 13/19 * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 13 23 RESULT: DCA # 26797 CONTAINS BTV SEROTYPE 13

24. SINGLE SEROTYPING 3, 12 & 14 3 26801 1214 DCA # 26801 MULTIPLEX A, B & C ABC 26801 24 12/14 3

25. DCA # 26801 MULTIPLEX HITS 3* 12/14 * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 12 25 RESULT: DCA # 26801 CONTAINS BTV SEROTYPE 12

26. SINGLE SEROTYPING 3, 4, 8, 9, 10 & 11 3 25394 1011 DCA # 25394 MULTIPLEX A, B & C ABC 25394 26 4 4 4 8,9,10,11 3 48 9 4389 10 11 ABC POS ITIVE CONTROL

27. DCA # 25394 MULTIPLEX HITS 3 4 8/9/10/11 * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 3, 4, 8, 9, 10 & 11 27 RESULT: DCA # 25394 CONTAINS BTV SEROTYPES 3, 4, 8, 9, 10 & 11

28. CONCLUSION MULTIPLEX PCR’S ARE MORE SENSITIVE THAN SINGLE SEROTYPING PCR’S ISOLATE CONSIDERED POSITIVEIF A SEROTYPE WAS IDENTIFIED BY BOTH MULTILEX AND SINGLE PCR MULTIPLEX PCR SCREENING DONE FIRST HITS CONFIRMED WITH LESS SENSTIVE SINGLE SEROTYPING PCR SEROTYPE 11 WAS NOT IDENTIFIABLE IN THE POSITIVE CONTROL, HOWEVER IT WAS FOUND IN ISOLATE 25394 FURTHER OPTIMIZATION AND SEQUENCING WILL BE CONDUCTED ON ALL 26 KNOWN BLUETONGUE VIRUS SEROTYPES 28

29. 29 ISOLATESEROTYPE(S) 265291 266501 267286 2680112 2679713 253943, 4, 8, 9, 10 & 11 GEOGRAPHICAL DISTRIBUTION OF BTV SEROTYPES IDENTIFIED