Table 2.

Yields of mutant viruses following electroporation of RNA into BHK-21 cells and two passages of virus in C6/36 cells

Site(s) mutatedVirusIFaVirus titerb (PFU/ml)Approx plaque size (mm)RT-PCRcATPase activityfHelicase activityf
MutantTemp (°C)d
V233++++(1.1 ± 0.1) × 106 4Yes++
V237++++(7.3 ± 0.8) × 106 4Yes
E 169 KSIE 173 V169–173 33e ++++(2.7 ± 0.2) × 106 3Yes
V169–173 37++++(2.2 ± 0.5) × 106 3Yes
E 179DD181 V179–181 33e +++(4.9 ± 1.0) × 105 1Yes
V179–181 37+++(1.4 ± 0.2) × 106 1Yes
R 184 KR 186 V184–186 33None detectedNo
V184–186 37None detectedNo
K199AVK199A 33None detectedNo
VK199A 37None detectedNo
M283FVM283F 33+++(4.7 ± 1.0) × 105 2Yes
VM283F 37None detectedNo
D 334 EE 336 V334–336 33++++(7.3 ± 1.1) × 102 1Yes
V334–336 37None detectedNo
R 376 KNGK 380 V376–380 33None detectedNo
V376–380 37None detectedNo
D 436GEE 439 V436–439 33None detectedNo
V436–439 37None detectedNo
R457A,R458AVR457A,R458A 33None detectedNo
VR457A,R458A 37None detectedNo
  • a Immunofluorescence (IF) in BHK-21 cells at 5 to 6 days postelectroporation. IF was scored as follows: −, no positive cells; +, 1 to 25% positive cells; ++, 25 to 50% positive cells; +++, 50 to 75% positive cells; ++++, 75 to 100% positive cells.

  • b Plaque titers after passaging in C6/36 are expressed as means ± one standard deviation. Each virus was derived at least twice from RNA transcripts; therefore, the result shown for each virus is the average of two or more experiments.

  • c Detection of product after RT-PCR. All positive samples retained the required mutation and had no other changes in the NS2B/3 genes.

  • d Temperature at which BHK-21 cells were incubated immediately after electroporation.

  • e Data for this virus are published in reference 37.

  • f ATPase and RNA helicase activities at 25°C. Activity was scored as no activity (−), parental activity (+), activity increased compared with parental activity (↑), or activity reduced compared with parental activity (↓) (Fig. 4B and Fig. 5D).