1. Thaw viral supernatant on ice (or in cold water). Be careful to keep thawed supernatant on ice at all times.
2. Spin supernatant at approx 2 K for 10 min at 4°C to pellet nonviral, cellular debris.
3. Carefully decant supernatant into 40 mL polyallomer ultracentrifuge tubes (Beckmann), which have been rinsed well with 70% ethanol and left to air-dry in the tissue-culture hood.
4. Spin at 20 K for 2 h at 4°C in a SW28 rotor to pellet viral particles.
5. Pour off supernatant, and aspirate well.
6. Allow the tubes to stand upright on ice for a few minutes.
7. Carefully resuspend the pellet in the supernatant left in the tube (usually about 50-100 pL) by gently pipeting up and down. Avoid making bubbles, since this can denature the viral proteins. Resuspension may take up to 2 h if the pellet is fairly sticky. It is convenient to leave the tubes in an ice bucket in the tissue-culture hood, and pipet the suspension approx once every 15 min.
8. Pool identical samples. To avoid repeated freeze-thawing, concentrated viral supernatant should be aliquoted into an appropriate volume convenient for injections (e.g., 20 pL) and stored at -70°C (see Note 8).
9. To determine the number of infectious virions/mL, concentrated viral supernatant stocks can then be titered by infecting either CEFs or the chemically transformed quail embryonic fibroblast cell line, QT6 (19). Viral titers are determined by infecting cells with serial dilutions of concentrated viral supernatant. Following an 18-48 h incubation, the cells are fixed using 3.5% paraformaldehyde, and the number of infected cells is determined by immunostaining using the monoclonal 3C2 anti-GAG antibody (18) (see Subheading 3.6.). Titers of concentrated viral stocks should be between 5 x 107 and 109 infectious virions/mL (see Note 9). Where possible, QT6 cells are used in preference to CEFs, since although they are initially infected as well as CEFs, the virus does not spread as rapidly from cell to cell, thus making it easier to identify clonal isolates. QT6 cells can be infected with viruses of all subtypes, except B and E.
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