Serum and CSF Samples Were Pretreated As Follows: 1 Ml of Serum Or CSF Was Centrifuged

E-Methods

Serum and CSF samples were pretreated as follows: 1 mL of serum or CSF was centrifuged at 10,000 g for 10 minutes at 4°C and the supernatant filtered using a 0.22 micron filter. To concentrate virus particles, 180 uL volumes of serum or CSF filtrates were diluted to 1.5 mL with 50mM Tris-HCl (pH 8.3) plus 50mM KCl buffer in 2.0 mL microcentrifuge tubes and centrifuged at 4°C at 78,200 g for 90 minutes. Supernatants were discarded and pellets were incubated for 20 minutes at room temperature in 75 µL lysis buffer containing: 50mM Tris-HCl (pH 8.4), 50mm KCL , 0.8mM EGTA, 20 mM DTT, 0.6% NP-40, and 0.1 mg/mL bovine serum albumin. Five microliters of lysed pellet solution was added to 20 µL of cDNA reaction mix (10 ng brome mosaic virus/BMV RNA [Promega, Madison, Wisconsin], 2 units recombinant RNase inhibitor [Promega], 80 ng BMVA primer (Integrated DNA Technologies, Coralville, IA), (ref. 7), 0.8mM EGTA, 2mM DTT, 50mM Tris-HCl (pH 8.3), 50mM KCl , 200 uM dNTP, 3 µM MgCl and 8.7 µg of activated DNA (Sigma-Aldrich, U.S.A.) and then overlaid with 40 µL of mineral oil and incubated at 37°C for 90 minutes, followed by a second incubation at 97°C for 10 minutes. cDNA of BMV RNA was amplified by nested PCR. In the first-round PCR, 5 uL cDNA and 20 uL PCR mastermix (50mm Tris-HCl [pH 8.3], 50mm KCl, 3mM MgCl, 200 µM of dNTP, 1 unit Amplitaq and 40ng of each outer primer BMV1A and BMV2, (ref. 7),) was overlaid with 40 µL of mineral oil. Mixtures were denatured for 5 minutes at 94°C, followed by 30 cycles of 94°C for one minute, 55°C for one minute and 72°C for one minute and, lastly, a 7-minute extension step at 72°C. For the second-round PCR, 2 uL of first-round PCR product was added to 23 uL of modified PCR mastermix (MgCl concentration was reduced to 1.5 mM and BMV3A and BMV4 inner primers, (ref. 7), were used in lieu of outer primers). Thirty additional amplification cycles were performed as in the first round, but the annealing temperature was increased from 55°C to 59°C. Recombinant Moloney murine leukemia virus reverse transcriptase (Promega) was run as a positive control. Negative lysis buffer controls were also included in each run. Ten microliters of second round PCR product was electrophoresed on ethidium bromide-containing 3% agarose gels. Bands were visualized under ultraviolet light and data was analyzed. The presence of sample reverse transcriptase was determined by the presence of a 79bp band, (figure 1).

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