Virus isolates and insect
Spodoptera littoralis was obtained from the insect rearing facility of the Faculty of Agriculture, Cairo University, Egypt. The larvae were kept at 26±1°C with 60% relative humidity and reared on the artificial diet previously described by Ivaldi-Sender (1974). Neonates and 4th larval instar were used for bioassay experiments and virus propagation, respectively. Virus used in this study is a field collected baculovirus isolate genus nucleopolyhedrovirus from infected dead larva collected from cabbage leaves. In addition, SpliNPV-AN1956 strain was used as a reference.
For virus propagation, the semi artificial diet described by Ivaldi-Sender (1974) was used. Semi-artificial diet was prepared using the following components: maize meal, Agar-Agar, brewer’s yeast, wheat germ, Ascorpic acid, and Nipagien (hydroxybenzoic acid methyl ester). After preparation and diet solidification, tiny cubes of the diet were put into each well of Raster boxes (50-well plates). About 10 μl of each virus suspension was prepared and added on each cube. The S. littoralis larvae (4th instar) were placed in every well, then plates were incubated at 26±1°C. One day later, larvae were transferred to virus-free semi artificial diet and reared till observation of viral infection symptoms (about a week post infection). Virus suspension was prepared according to the polyhedral counting (OBs/ml) for each isolate.
Virus occlusion bodies’ counting
Viral OBs’ purification was carried out according to Boughton et al. (1999). Briefly, infected S. littoralis larvae were collected and homogenized using 0.1% SDS, followed by filtration using a piece of cotton and filter paper. After centrifugation of the suspension, the pellets were re-suspended in 0.5 M NaCl and the final pellet contains OBs was re-suspended in suitable volume of ddH2O. The diluted OBs were counted under the dark field, using inverted microscope with ×200 magnification (Axio-VertA1, Zeiss, Germany). Counting was performed using a Petroff-Hauser counting chamber (depth 0.01 mm, Hausser Scientific). The purified virus OBs were kept frozen at −20°C, subsequently, examined under light microscope as well as electron microscope.
In order to examine the viral OBs under a transmission electron microscope (TEM), the purified OB suspensions was loaded on carbon coated grib, followed by staining with 2% phosphor tangistic acid before examination under TEM (JEOL model JEM-1200EX II). The sample images were visualized and photographed at different magnification illustrated. The TEM photos provided by a scale were used to measure the sizes of the OBs.
Viral DNA purification
The SpliNPV genomic DNA was isolated from the purified viral OBs according to the method described by Boughton et al. (1999). Briefly, the purified OBs were dissolved, using 1M of Na2CO3 to release virions (ODV). Subsequently, ODV were treated with 10% (w/w) SDS, followed by adding 1%. Proteinase K allow genomic DNA to be released. The nucleic acids were collected from cells debris by washing 2 times, using TE-buffer saturated phenol/chloroform 1:1 (v/v). The viral genomic DNA was precipitated using 96% ethanol and 1:10 volume of 3M NaAc pH 5.2. After washing with 70% ethanol, the purified genomic DNA was eluted in suitable amount of ddH2O.
Digestion of viral DNA
For comparison of genome pattern of the SpliNPV isolate with the reference strain, restriction digestion was performed, using HindIII, PstI, and ScaI endonucleases at 37°C for 2. Electrophoresis was performed for the digested samples overnight using 0.8 % agarose in 1X TAE buffer (Boughton et al., 1999).
PCR amplification and sequencing of lef-5 gene
In order to partially amplify lef-5 gene, one set of specific primer pair were used. Primers used denoted LEF-5 (ssr1) 5′-AGTCATAAAATCATCGTCGGCG-3′ and LEF-5 (ssr1) 5′-GATTCTCACACGGCGCTCTC-3′. The PCR reactions were performed in a total volume of 25 μl containing the following components: 12.5 μl of EmeraldAmp® GT PCR Master Mix - Takara Bio, 1 μl of each forward and reverse primers, 2 μl of viral genomic DNA template (500ng). Volume was completed to 25 μl using autoclaved ddH2O. The PCR program was initiated at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 1 min, 60°C for 1 min and primer extension at 72°C for 45 s. The primer extension was completed at 72 °C for 7 min. PCR-amplicon was electrophoresed in a 1% agarose gel prepared in 1X TAE buffer. The PCR amplicon was visualized using UV-transilluminator. The PCR fragments were purified from agarose gel using Qiaquick PCR purification kit (Qiagene, Germany). Sequence of the purified fragments was performed using Sanger sequence.
The partial lef-5 gene sequence was subjected for alignment with the published ones, using Blast search data base of the National Centre for Biotechnology Information (NCBI). Analysis of the deduced amino acid sequence was carried out, using EditSeq-DNAstar Inc., sequence analysis software, Windows 32 Edit Seq 4.00 (1989–1999). The multiple sequence alignment analysis and the phylogenetic tree were achieved, using Clustal Omega for multiple alignment analysis (The EMBL-EBI search and sequence analysis tools APIs in 2019).
The virulence of both examined and reference isolates was determined, using median lethal concentration (LC50) and median survival time (ST50). LC50 was determined by exposure of the 1st instar S. littoralis larvae to serial dilutions of virus OBs suspensions of 1 × 103, 5 × 103, 1 × 104, 5 × 104, 1 × 105, and 5 × 105 (OBs/ml), in addition to diet mixed with ddH2O instead of virus suspension as a negative control. Each concentration from both isolates was replicated 5 times where each replicate contained 20 larvae. For estimation of LC50, the total dead larvae after 7 days of infection were counted. In order to determine viruses’ speed of kill, the ST50 was estimated.by inoculating 30 individuals of S. littoralis (5 days old) with 3 replicates and calculated LC85 for each tested viral isolate. Control plates were mixed with water instead of virus suspension. Mortality was observed 12-h intervals starting at the 3rd after infection till the 10th day or larval death.
The LC50 and slopes of tested viral isolates were estimated using EPA Probit analysis program (Version 1.5) (Robertson and Preisler, 1992). Also, the ST50 was estimated. Data analysis was performed, using the Kaplan-Meier survival time estimator analysis (Kaplan-Meier, 1958).