Sindbis and Middelburg Old World Alphaviruses Associated with Neurologic Disease in Horses, South Africa

Old World alphaviruses were identified in 52 of 623 horses with febrile or neurologic disease in South Africa. Five of 8 Sindbis virus infections were mild; 2 of 3 fatal cases involved co-infections. Of 44 Middelburg virus infections, 28 caused neurologic disease; 12 were fatal. Middelburg virus likely has zoonotic potential.

The first round and nested PCR primers that were based on a highly conserved region of the nsP4 gene were prepared as described previously (1). First-round PCR was performed by using the Titan one tube reverse transcription (RT)-PCR system (Roche). Each reaction contained 10 μL of RNA, 10 μL 10× reaction buffer, 1 μL of dNTP mix (10 mmol/L), 2 μL of 10 pmol of each primer (Alpha1+; Alpha1-), 2.5 μL Dithiothreitol solution (100 mmol/L), 0.25 μL RNase inhibitor (40 U/μL), and 1 μL of the Titan enzyme mix. The final volume was made up to 50 μL with distilled water. Cycling conditions were 50°C for 30 min, 94°C for 2 min, (94°C for 10 s, 52°C for 30 s, 68°C for 1 min) × 35 cycles, and 68°C for 7 min. Differentiation of MIDV and SINV was performed on first-round RT-PCR products by real-time PCR by using the Lightcycler TaqMan Master kit (Roche): 2 μL of RT-PCR product was used with 1 μL of 20 pmol of each primer (Alpha2+; Alpha2-), 0.2 μL of 10 pmol probe each for SINV and MIDV, 4 μL of FastStart enzyme mixture to a final volume of 20 μL. The following program was followed on a Lightcycler 2 machine (Roche): preincubation at 95°C for 10 min (95°C for 10 s, 52°C for 1 min, 72°C for 1 s) for 45 cycles. Qualitative analysis that used different channels could distinguish between SINV and MIDV.

SINV E-Protein RT-PCR
Titan one tube RT-PCR system (Roche) was used for first-round PCR by using primer SIN8136EF (5 TCGTCAGCATACGACATGGAG 3) and A2 (3). PCR amplification began at 94°C for 2 min and 40 cycles of 94°C for 10 s, 52°C for 30 s and 72°C for 1 min with a final elongation step of 72°C for 7 min.
Expand High Fidelity PLUS PCR system (Roche) was used for the semi-nested reaction. A total of 5μL of first-round product was used with primers SIN8136EF and SIN8787ER (5GTATCCAAACTGGGCGGAAGT 3). The following cycling program was used: 94°C for 2 min and 10 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min; 25 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min + 10 s/cycle with a final elongation step of 72°C for 7 min.

MIDV E-Protein RT-PCR
Titan one tube RT-PCR system (Roche) was used for first-round PCR with primer MID10475E+ (5 GGTGCACGTTCCATATACCC 3) and MID11045E-(5 TCCCAATAGCAATCACCACA 3). Cycling began at 94°C for 2 min and 40 cycles of 94°C for 30 s, 48°C for 45 s, and 72°C for 1 min with a final extension of 72°C for 7 min.
Nested PCR was performed with Expand High Fidelity PLUS PCR system (Roche) by using 2μL of first round template, 5μL Q-solution (QIAGEN, Valencia, CA, USA), and primers MID10543EN+ (5 TGAACCACAAGGCTCCTTTC 3) and MID10911EN-(5 CACTTTGCTGTGCAAGTGGT 3). Cycling commenced at 94°C for 2 min and 40 cycles of 94°C for 30 s, 50°C for 45 s and 72°C for 1 min, with a final elongation step of 72°C for 7 min.