The KOUTV strain utilized in this experiment was the KOUTV DAK Ar D 5443 received from Robert B. Tesh (CBEID-UTMB). Virus was propagated by inoculating 100 μL of viral stock into the T-75 flask of confluent Vero cells. To determine titer, a plaque assay was developed and standard curves created for quantitative reverse transcription polymerase chain reaction (qRT-PCR) as previously described.16 (link) Briefly, we utilized a SuperScript III One-step qRT-PCR kit (Invitrogen, Carlsbad, CA) with the primers and probes given in Table 1 .
The presence of KOUTV viral RNA was detected via qRT-PCR using the following protocol: RT step (1 cycle) 48° C for 2 minutes and 95 °C for 2 minutes, amplification and data recording step (40 cycles) 95 °C for 15 seconds and 60 °C for 30 seconds. Primers were designed and obtained via Integrated DNA technologies with 5′-FAM fluorophore and 3′-Black-Hole quencher. Prior to offering virus to mosquitoes, virus was propagated into Vero cells. Viral standard curves and concentrations were obtained via plaque assay as described previously before the beginning of the experiment.16 (link)
The presence of KOUTV viral RNA was detected via qRT-PCR using the following protocol: RT step (1 cycle) 48° C for 2 minutes and 95 °C for 2 minutes, amplification and data recording step (40 cycles) 95 °C for 15 seconds and 60 °C for 30 seconds. Primers were designed and obtained via Integrated DNA technologies with 5′-FAM fluorophore and 3′-Black-Hole quencher. Prior to offering virus to mosquitoes, virus was propagated into Vero cells. Viral standard curves and concentrations were obtained via plaque assay as described previously before the beginning of the experiment.16 (link)