Source of insects and virus
Corn earworm larvae used to start a laboratory colony of healthy H. zea were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously .
Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation .
Injection of adults
Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand . The female moths were divided into four dose groups, and 9 or 10 insects were infected with Hz-2V at one of the following concentrations of 2 × 105, 2 × 106, 2 × 107, and 2 × 108 TCID50 units.
Tn368 cells were cultured as per Burand & Lu  and 100 ul of cell culture medium containing 8 × 104 Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID50 calculated .
DNA extraction and purification of viral DNA
DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min.
Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu .
PCR amplification of viral DNA sequences
Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set (P4-1, 5'-GCACGATTCGTAATGTTC-3'; and P4-2, 5'-GCACACCTATCAATCACC-3') was designed to amplify a 434 bp sequence of the Hz-2V genome . PCR reactions using P4-1 and P4-2 primers were brought to a final volume of 20 ul using the Bioneer AccuPower® PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2 and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V.
The second set of primers (P4-3, 5'-GCTGTGCTGTACAAGTGC-3'; and P4-4, 5'-CCCTTGACGATCCCTTTTG-3') was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer (100 mM Tris-HCl pH 8.0, 500 mM KCL pH 8.3, and 25 mM MgCL2), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP (2 mM dATP, dCTP, dGTP, 1.3 mM dTTP, and 0.7 mM alkali labile DIG-11 dUTP pH 7.0), and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit.
Both PCR reactions for amplification of the viral DNA in tissue samples and for the production of the viral DNA probe consisted of 30 cycles of a DNA denaturation step at 95°C for 1 min., a primer annealing step for 1 min. at 55°C, and a 1 min. primer extension step at 72°C.
Detection of a viral DNA sequence by slot blotting
To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA (10 mM, pH 8.2) at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer (0.6 M NaCl, 60 mM Na citrate pH 7.0) in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer (5X SSC, 0.1% (w/v) N-laurylsarcosine, 1% (w/v) Na2-Dodecylsulfate, 2% Blocking reagent (Boehringer-Manheim), and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim).
Analysis of PCR products by agarose gel electrophoresis
In order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.