A comparison of techniques for detecting Invertebrate iridescent virus 6

The aim of this study was to compare the sensitivity and precision of vario us methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo s uppressalis, and to apply these techniques to the detection of covert infec tions in the wax moth, Galleria mellonella. The relationship between the vi rus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID50 assays using 12 different cell l ines indicated that two Drosophila lines, DL2 and DR1, had the highest susc eptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID50 values for IIV-6 in S podoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-6 4 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of I particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the ins ects that survived to adulthood, between 5.8 and 75% caused patent infectio ns when injected into G. mellonella larvae, indicating that they were cover tly infected. A PCR technique resulted in 95% detection at 1000 virus parti cles per insect. Of the insects that proved positive for covert infection b y insect bioassay, 41% also proved positive by PCR analysis. It is conclude d that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay. (C) 2001 Elsev ier Science B.V. All rights reserved.