title: A comparison of techniques for detecting Invertebrate iridescent virus 6.
abstract: The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 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 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded 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.
authors.affiliation: ECOSUR, Apdo Postal 36, Tapachula 30700, Chiapas, Mexico.
authors.forename: M
authors.initials: M
authors.lastname: Constantino
authors.forename: P
authors.initials: P
authors.lastname: Christian
authors.forename: C F
authors.initials: CF
authors.lastname: Marina
authors.forename: T
authors.initials: T
authors.lastname: Williams
mesh_terms: D000330:Aedes / Q000166:cytology / Q000821:virology*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D004330:Drosophila / Q000821:virology; D007304:Insect Viruses / Q000302:isolation & purification* / Q000648:ultrastructure; D007497:Iridoviridae / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D007814:Larva / Q000821:virology; D008855:Microscopy, Electron, Scanning; D009036:Moths / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D018411:Spodoptera / Q000166:cytology / Q000821:virology*; D014771:Virion / Q000254:growth & development; D014776:Virus Cultivation