PFGE and in-gel hybridization enabled estimation of chromosome sizes in different strains and testing for the presence of the virus genome. PFGE and in-gel hybridization were performed as previously described with modifications as follows (44). Cells (8.7 × 107) in exponential growth phase were harvested by centrifugation (8000g for 20 min), resuspended in 150 μl of TE buffer [10 mM tris-HCl and 125 mM EDTA (pH 8)], and then embedded in plugs by mixing with an equal volume of molten low–melting point agarose (1% in TE buffer precooled to 45°C). After setting, the cells were lysed in the agarose plugs by immersing the plugs in proteinase K buffer [10 mM tris HCl (pH 8), 0.5 M EDTA (pH 8), 1% lauroyl sarcosinate, and proteinase K (1 mg ml−1) (final concentration)] at 37°C for 24 hours under constant agitation. The plugs were then washed three times in EDTA solution [0.5 M (pH 8)] at 37°C for 2 hours under constant agitation and then stored in EDTA solution at 4°C. PFGE was performed in 0.8% agarose gels in 0.5× tris-borate EDTA buffer [44.5 mM tris, 44.5 mM boric acid, and 1 mM EDTA (pH 8)] with 2 mm of plug from each sample loaded into the wells. Electrophoresis was run in the CHEF-DR III (Bio-Rad) system at 14°C and 3 V cm−1 with 120° pulse angle for 45 hours with a switch time of 90 s, followed by 27 hours at a switch time of 140 s. After PFGE, DNA was visualized by immersing the gel in a solution of ethidium bromide. The gel was moved to a flat-bottomed dish, and enough 0.4 M NaOH was added to cover the gel for chemical denaturation of the DNA by incubation for 30 min at room temperature with agitation. Then, the gel was washed three times for 10 min in a 6× SSC solution [20× SSC, 3 M NaCl, and 0.3 M Na3citrate (pH 7)]. Last, the gel was dehydrated under vacuum (GD2000, Hoefer Inc., Richmond, CA, USA).

PCR (GoTaq, Promega) was used to amplify specific regions of the SOC, Big Outlier Chromosome (BOC), OmV2, and the 18S rRNA gene (see table S3 for primer sequences) for use as DNA probes with the following conditions: initial denaturation for 2 min at 94°C; 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 45 s at 72°C; and final extension for 5 min at 72°C. The PCR product was cleaned with Wizard (Promega) SV Gel and PCR Clean-Up System. The amplicons were randomly labeled with [α-32P]cytidine triphosphate (PerkinElmer catalog no. BLU008H250UC) according to the manufacturer’s instructions (Prime-a-Gene kit, Promega, catalog no. U1100) for use as DNA probes. Dried gels were equilibrated in sufficient hybridization buffer [6× SSC, 5× Denhardt’s solution, 0.1% (v) SDS, and tRNA (10 μg ml−1)] to cover the gels for 3 hours at 65°C with constant agitation, after which radiolabeled probes were added and hybridized overnight at 65°C with constant agitation. After hybridization, the gel was subjected to a series of washes with constant agitation: three washes in 6× SSC buffer at room temperature for 30 min, one wash in 6× SSC buffer at 65°C for 1.5 min, followed by a final wash in 3× SSC buffer at room temperature for 5 min. The gel was sealed in a transparent plastic sleeve and then exposed to radiographic film.

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