3.8 Verification
of Virus Titers
The titers of replication-proficient virus can be verified by standard
plaque assay methods [35]. In contrast, as replication-deficient
particles do not generate plaques, methods for titer determination
are limited although indirect titer estimations are possible by calculating the number of infected cells visualized by reporter gene
expression.
1. BHK-21 (or other) cells are cultured to a defined confluency
on 6- or 12-well plates or on sterile coverslips before infection
with serial dilutions (e.g., fivefold dilutions in the range
expected to give 20–50 positive cells per microscope field) of
virus stocks expressing green fluorescent protein (GFP) or
β-galactosidase.
2. Cells expressing the reporter gene (fluorescent or stained cells,
see below) are counted after 24 h incubation at 37
C. Certain
SFV mutant vectors may provide a weak signal at 24 h postinfection and should be incubated for 48 h to reach optimal
expression levels.
3.8.1 GFP Detection
1. The number of GFP-positive cells are counted applying fluorescence microscopy.
2. The approximate titers are estimated as infectious particles/mL
based on the number of GFP-positive cells per well, by taking
into account the virus dilution.
3.8.2 X-gal Staining
1. SFV-infected cells are washed with PBS, fixed in cold methanol
(99.8%) at À20
C for 5 min and washed again three times
with PBS.
2. Cells are stained for at least 2 h in X-gal staining solution at
37
C or at room temperature.
3. The number of β-galactosidase (blue) positive cells are counted
applying light microscopy.
4. Approximate titers are estimated as described for GFP detection above.
For comparison of GFP and X-gal methods, please see Note 4.
3.8.3 Immunofluorescence
If antibodies are available against the recombinant protein itself, or
against tags engineered in the vector construct, immunofluorescence methods can be applied for titer determination.
1. SFV-infected cells are cultured on sterile coverslips, rinsed
twice with PBS and fixed for 6 min at À20
C in methanol.
2. Coverslips are washed three times in PBS and incubated for
30 min at room temperature in PBS containing 0.5% gelatin
and 0.25% BSA to prevent nonspecific binding.
Alphavirus-Based Antigen Preparation
73
of Virus Titers
The titers of replication-proficient virus can be verified by standard
plaque assay methods [35]. In contrast, as replication-deficient
particles do not generate plaques, methods for titer determination
are limited although indirect titer estimations are possible by calculating the number of infected cells visualized by reporter gene
expression.
1. BHK-21 (or other) cells are cultured to a defined confluency
on 6- or 12-well plates or on sterile coverslips before infection
with serial dilutions (e.g., fivefold dilutions in the range
expected to give 20–50 positive cells per microscope field) of
virus stocks expressing green fluorescent protein (GFP) or
β-galactosidase.
2. Cells expressing the reporter gene (fluorescent or stained cells,
see below) are counted after 24 h incubation at 37
C. Certain
SFV mutant vectors may provide a weak signal at 24 h postinfection and should be incubated for 48 h to reach optimal
expression levels.
3.8.1 GFP Detection
1. The number of GFP-positive cells are counted applying fluorescence microscopy.
2. The approximate titers are estimated as infectious particles/mL
based on the number of GFP-positive cells per well, by taking
into account the virus dilution.
3.8.2 X-gal Staining
1. SFV-infected cells are washed with PBS, fixed in cold methanol
(99.8%) at À20
C for 5 min and washed again three times
with PBS.
2. Cells are stained for at least 2 h in X-gal staining solution at
37
C or at room temperature.
3. The number of β-galactosidase (blue) positive cells are counted
applying light microscopy.
4. Approximate titers are estimated as described for GFP detection above.
For comparison of GFP and X-gal methods, please see Note 4.
3.8.3 Immunofluorescence
If antibodies are available against the recombinant protein itself, or
against tags engineered in the vector construct, immunofluorescence methods can be applied for titer determination.
1. SFV-infected cells are cultured on sterile coverslips, rinsed
twice with PBS and fixed for 6 min at À20
C in methanol.
2. Coverslips are washed three times in PBS and incubated for
30 min at room temperature in PBS containing 0.5% gelatin
and 0.25% BSA to prevent nonspecific binding.
Alphavirus-Based Antigen Preparation
73