A comparison of techniques for detecting Invertebrate iridescent virus 6.

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.

A possible iridovirus in erythrocytes of Bufo marinus in Costa Rica.

Icosahedral viral particles were found in the cytoplasm of erythrocytes and splenic reticular cells of a marine toad (Bufo marinus) collected from Costa Rica. Capsids had a maximum diameter of 312 nm and a spherical core with biphasic electron density. Viruses in erythrocytes were associated with cytoplasmic assembly areas and vacuoles in cytoplasm. Nuclei had finely granular material of decreased electron density located centrally, but contained no viral particles. A group of unenveloped viral particles was seen extracellularly in a splenic vessel. The virus was consistent with an iridovirus. In a blood smear stained with Giemsa round basophilic bodies with average diameters of 1.70 microns and morphologically similar to Pirhemocyton sp. were seen in the cytoplasm of erythrocytes and occasionally in the cytoplasm of monocytes or extracellularly. Erythrocytes containing these bodies had vacuoles and irregular pale-staining areas in the cytoplasm and pale-staining areas in the nucleus. These changes corresponded to the viral particles, assembly areas, vacuoles and nuclear changes at the ultrastructural level.