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 1

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

PRIMER DESIGN vComp.svg/2000px-Primers_RevComp.svg.png 3

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

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

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

BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR DIFFERENTIAL PRIMER CONCENTRATIONS µM µM µM µM µM 95°C 55°C 72°C 4°C 1 Min15 sec 12 sec1 Min 35 Cycles 7

BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR MULTIPLEX PRIMER CONCENTRATIONS µM µM µM µM µM 95°C 55°C 72°C 4°C 1 Min15 sec 90sec1 Min 35 Cycles

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

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

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

BOTTLE B AND C MULTIPLEX SEROTYPING PCR all primers 0.6 µM CB 12

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

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 µM µM µM µM µm MULTIPLEX PRIMER CONCENTRATIONS BOTTLE B µM µM µM µM µM MULTIPLEX PRIMER CONCENTRATIONS BOTTLE C µM µM µM µM µM ABC 14

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

DCA # MULTIPLEX A, B & C ABC SINGLE SEROTYPING 1, 3, 4, 6, 13 & ,4,6 3 13/

DCA # MULTIPLEX HITS /19* * SPECULATIVE HITS HITS CONFIRMED WITH SINGLE SEROTYPING 1 17 RESULT: DCA # CONTAINS BTV SEROTYPE 1

DCA # MULTIPLEX A, B & C ABC SINGLE SEROTYPING 1, 3, 13 & /

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

DCA # MULTIPLEX A, B & C ABC SINGLE SEROTYPING 4 & /

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

SINGLE SEROTYPING 3, 5, 7, 13 & DCA # MULTIPLEX A, B & C ABC /6 13/19 5,7 3

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

SINGLE SEROTYPING 3, 12 & DCA # MULTIPLEX A, B & C ABC /14 3

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

SINGLE SEROTYPING 3, 4, 8, 9, 10 & DCA # MULTIPLEX A, B & C ABC ,9,10, ABC POS ITIVE CONTROL

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

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 FURTHER OPTIMIZATION AND SEQUENCING WILL BE CONDUCTED ON ALL 26 KNOWN BLUETONGUE VIRUS SEROTYPES 28

29 ISOLATESEROTYPE(S) , 4, 8, 9, 10 & 11 GEOGRAPHICAL DISTRIBUTION OF BTV SEROTYPES IDENTIFIED