Collection and extraction of baculovirus
The NPV was isolated from the dead larvae of E. chrysorrhoea, collected from apricot trees, in the Ladakh region of India. To release occlusion bodies (OBs), the diseased larvae were homogenised, using a sterile pestle and mortar for 4 min with 5 ml of distilled water. The suspension was filtered twice through a double-layered muslin cloth and then the filtrate was centrifuged (Remi, cC24 BL, India) at 500 rpm for 1 min to remove the larger particles. The supernatant was suspended in (5 ml) distilled water and centrifuged at 5000 rpm for 20 min to collect the pellet containing polyhedra. The pellet containing OBs was re-suspended in (5 ml) distilled water and stored at 5 °C. The polyhedral occlusion bodies were counted using a haemocytometer.
Electron microscopy—morphology
The morphological studies of the extracted OBs were carried out under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The purified OB suspensions were taken in vials, fixed in 2.5% (v/v) glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4 °C. The samples were then post-fixed in 2% (v/v) aqueous osmium tetroxide (OsO4) prepared in 0.1 M phosphate buffer (pH 7.4) for 1 h and dehydrated in graded ethanol series (Martins et al., 2005). The samples were finally dried to a critical drying point. Stubs with double-sided conductivity carbon adhesive tape and sputter coated with gold for 20 s, using an automated sputter coater (Model: EMITEC - SC 7620), were used for sample mounting. The coated samples were examined directly under a SEM (Quanta 250, FEI, Netherlands) at 10 kV with a spot size of 3.5 and a pressure of 60 Pa. The sample images were visualised and photographed at × 40,000 magnification. The amplified photographs developed by a scale were used to measure the sizes of the OBs.
For TEM studies, the pellets of OBs were initially fixed in 2.5% (v/v) glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at 4 °C and again fixed in 0.5% (v/v) aqueous osmium tetroxide in the same buffer for 2 h. After the post-fixation, samples were dehydrated in a series of graded alcohol; the dehydrated sample was mounted on 300 mesh carbon-coated copper grid. The sample was stained by saturated aqueous uranyl acetate and counterstained with lead citrate and viewed under TECNAI 120 Kv TEM (FEI, Netherlands). The sizes of the OBs and nucleocapsids were measured directly from the amplified photographs using a precision ruler and compared to the magnification of the photograph.
Bioassay
Median lethal concentration (LC50) of Euproctis NPV to second instar larvae was estimated by a leaf disc bioassay method (Magholi et al., 2014) with few modifications. Viral suspensions of 102, 104, 106, and 108 OBs/ml were prepared in aqueous (0.05%) Tween 20 (v/v). Ten microlitres of viral suspension was spread on apricot leaf discs (2.0 cm diameter), air dried, and individually placed inside 12-well tissue culture plates (2.5 cm dia.). Each plate constituted an independent replication, and three plates were used per viral suspension, constituting 50 insects per treatment. Second instar E. chrysorrhoea larvae starved for about 6 h were released individually into the wells which were then covered with a lid. The larvae consumed the entire diet within 12 h, then were transferred to plastic containers containing fresh apricot leaves and maintained at 26 ± 2 °C and 60–70% relative humidity. In control, larvae were allowed to feed on leaves treated with aqueous (0.05%) Tween 20. Larval mortality rates were recorded at daily intervals. Mortality due to viral infection was recorded up to 9 days post inoculation.
Statistical analysis
Bioassay data were analysed, using the Probit analysis (Finney, 1971) to record the lethal concentration of virus required to cause 50% mortality (LC50). The data was analysed by analysis of variance (ANOVA), using SPSS (v.15.1; SPSS Inc., Chicago, IL). Mortality (%) was corrected by the following equation (Abbott 1925):
$$ M\left(\%\right)\frac{\left(t-c\right)}{\left(100-c\right)}\times 100 $$
where M = corrected mortality, c = mortality (%) in controls, and t = mortality (%) in treatments.