Afiltration devices (Pall) and processed for PFGE as previously described [24]. PFGE

Afiltration devices (Pall) and processed for PFGE as previously described [24]. PFGE was carried out using a CHEFDR II PFGE system (Bio-Rad) in Tris-Borate EDTA (TBE) buffer for 18 h with switch time ramping linearly from 1 to 12 s. DNA molecular weight 22948146 markers (MidRange I and Lambda Ladder; New England Biolabs) and mass standards (High DNA Mass Ladder, Invitrogen) were run on all gels. Gels were stained overnight at 4uC with SYTO 60 (Invitrogen), then visualized and analyzed with the Odyssey Infrared Imaging System (Li-Cor Biosciences).Ti, Beckman Coulter) in an Optima XL-80K ultracentrifuge (Beckman Coulter). Fractions of ,500 ml were collected top down from the gradient using a fraction collector (Auto Densi-Flow, Labconco) on low speed. Density of the fractions was determined gravimetrically and viruses were enumerated in each fraction using epifluorescence microscopy [25] with the stain SYBR Gold (Invitrogen). Assuming an average DNA content of 55 ag per virus [26], the volume of fraction required to obtain 100 ng of viral DNA was prepared for viral genome fingerprinting. A viscous whitish substance was observed in the completed CsCl gradient at densities .1.4 g ml21. The distribution of genome sizes in fractions was the same in all fractions from this zone and similar to the unfractionated sample. Under the assumption that the viruses in this zone were aggregated or adsorbed to the unknown whitish substance, an attempt was made to desorb the viruses. The relevant fractions were pooled and Tween-80 (Fisher) was added at a final concentration of 1 followed by MedChemExpress SC 1 sonication of the Triptorelin chemical information sample for 3 minutes in a sonicator bath (Branson). The treated sample was then fractionated in a second continuous CsCl gradient. A fraction from the continuous CsCl gradient was selected for further separation of viruses by strong anion-exchange chromatography [23]. A BioLogic HR Workstation (Bio-Rad) equipped with a 1-ml sample injector, gradient mixer, fraction collector, and UV and conductivity meters was used to run a step gradient through an UNO Q1 strong anion-exchange chromatography column (Bio-Rad). The starting buffer (20 mM Tris-HCl, pH 7.8) and elution buffer (20 mM Tris HCl, 1 M sodium chloride, pH 7.8) for chromatography were prepared with ultrapure water (NANOPure), autoclaved, and filtered through 0.22 mm pore-size filters. The remaining portion of the selected CsCl gradient fraction that had not been used for viral genome fingerprinting was exchanged into the chromatography starting buffer with a Centricon-20 centrifugal ultrafiltration device with a 100 kDa NMWCO filter (Millipore) and recovered at a final volume of ,1.1 ml. The UNO Q1 chromatography column was equilibrated sequentially with 7 ml of starting buffer, 7 ml of elution buffer, and 7 ml of starting buffer at 1 ml min21. The sample was then loaded onto the column and a step gradient was run with 1 steps of increasing elution buffer between 26 and 42 elution buffer at 0.5 ml min21, with collection of 8 ml fractions per step. For each fraction, 300 ml was used for viral genome fingerprinting and the remaining volume was stored at 4uC. A fraction from this gradient was then selected for analysis with transmission electron microscopy (TEM), shotgun clone library construction, and sequencing.Transmission Electron MicroscopyThe morphological diversity of viruses in the selected fraction was investigated with TEM. An air-driven ultracentrifuge (Airfuge CLS, Beckman) was used to deposi.Afiltration devices (Pall) and processed for PFGE as previously described [24]. PFGE was carried out using a CHEFDR II PFGE system (Bio-Rad) in Tris-Borate EDTA (TBE) buffer for 18 h with switch time ramping linearly from 1 to 12 s. DNA molecular weight 22948146 markers (MidRange I and Lambda Ladder; New England Biolabs) and mass standards (High DNA Mass Ladder, Invitrogen) were run on all gels. Gels were stained overnight at 4uC with SYTO 60 (Invitrogen), then visualized and analyzed with the Odyssey Infrared Imaging System (Li-Cor Biosciences).Ti, Beckman Coulter) in an Optima XL-80K ultracentrifuge (Beckman Coulter). Fractions of ,500 ml were collected top down from the gradient using a fraction collector (Auto Densi-Flow, Labconco) on low speed. Density of the fractions was determined gravimetrically and viruses were enumerated in each fraction using epifluorescence microscopy [25] with the stain SYBR Gold (Invitrogen). Assuming an average DNA content of 55 ag per virus [26], the volume of fraction required to obtain 100 ng of viral DNA was prepared for viral genome fingerprinting. A viscous whitish substance was observed in the completed CsCl gradient at densities .1.4 g ml21. The distribution of genome sizes in fractions was the same in all fractions from this zone and similar to the unfractionated sample. Under the assumption that the viruses in this zone were aggregated or adsorbed to the unknown whitish substance, an attempt was made to desorb the viruses. The relevant fractions were pooled and Tween-80 (Fisher) was added at a final concentration of 1 followed by sonication of the sample for 3 minutes in a sonicator bath (Branson). The treated sample was then fractionated in a second continuous CsCl gradient. A fraction from the continuous CsCl gradient was selected for further separation of viruses by strong anion-exchange chromatography [23]. A BioLogic HR Workstation (Bio-Rad) equipped with a 1-ml sample injector, gradient mixer, fraction collector, and UV and conductivity meters was used to run a step gradient through an UNO Q1 strong anion-exchange chromatography column (Bio-Rad). The starting buffer (20 mM Tris-HCl, pH 7.8) and elution buffer (20 mM Tris HCl, 1 M sodium chloride, pH 7.8) for chromatography were prepared with ultrapure water (NANOPure), autoclaved, and filtered through 0.22 mm pore-size filters. The remaining portion of the selected CsCl gradient fraction that had not been used for viral genome fingerprinting was exchanged into the chromatography starting buffer with a Centricon-20 centrifugal ultrafiltration device with a 100 kDa NMWCO filter (Millipore) and recovered at a final volume of ,1.1 ml. The UNO Q1 chromatography column was equilibrated sequentially with 7 ml of starting buffer, 7 ml of elution buffer, and 7 ml of starting buffer at 1 ml min21. The sample was then loaded onto the column and a step gradient was run with 1 steps of increasing elution buffer between 26 and 42 elution buffer at 0.5 ml min21, with collection of 8 ml fractions per step. For each fraction, 300 ml was used for viral genome fingerprinting and the remaining volume was stored at 4uC. A fraction from this gradient was then selected for analysis with transmission electron microscopy (TEM), shotgun clone library construction, and sequencing.Transmission Electron MicroscopyThe morphological diversity of viruses in the selected fraction was investigated with TEM. An air-driven ultracentrifuge (Airfuge CLS, Beckman) was used to deposi.