Virological methods

Methods

3.4.9. Flow cytometry

For analyzation of the EGFP AAV replicon stable cell line, EGFP expression was measured via Fluorescence-based flow cytometry assay. Typically, AAVG clones were seeded 1:4 in duplicates in a new 6-well plate and infected 24 h later or mock treated. After 3 days cells were harvested by digestion with Trypsin/EDTA and washed with PBS. Cell pellets were re-suspended in 0.8 - 2 mL PBS and measurement was done using the BD FACS Canto™ II cytometer.

Methods

followed before the suspension was centrifuged again at 6.000 x g at RT for 10 min. Sn was collected for further processing.

3.5.3. Preparation of viral inocula

For preparation of low titer virus inocula optional amount and size of cell culture dishes with the appropriate cells grown to 90% confluency were infected with either a reconstituted virus BAC or a previous virus stock. Ad were propagated in 293A cells upon infection at a MOI of 1.

Harvesting of lysed cells and Sn followed 1 day after complete cytopathic effect was observed.

The pooled suspension was centrifuged at 6.000 x g at 4°C for 15 min. Except of 2 mL the Sn was collected, and an aliquot was stored in -80°C for safety. The cell pellet was re-suspended in the remaining 2 mL Sp. Then three cycles of freezing (5 min in liquid nitrogen) and thawing (10 min at 37°C) followed before the suspension was centrifuged again at 6.000 x g at 4°C for 15 min. Viruses in the Supernatants were harvested and Aliquots of 250 µL were stored at -80°C. Virus titer was analyzed by endpoint titration (chapter 3.5.5).

For propagating HSV, Vero cells were infected at MOI 0.01 and cell-virus suspension was collected 4 d p.i., harvested and frozen at -20°C. After a thawing process to 37°C, suspension was vortexed for 15 sec and centrifuged at 6.000 x g at 4°C for 15 min. Then the Supernatants were aliquoted using a volume of 100 µL and stored at -80°C.

3.5.4. Preparation of high-titer virus stocks

Large scale adenovirus stocks were purified using the caesium chloride (CsCl) purification method. To do so, first a seed stock of the virus had to be generated to be able to propagate a bulk stock. Therefore 150 µl of either a previous virus stock or of the 2^nd lysate after BAC reconstitution (chapter 3.5.2) were used to infect 293A cells grown to a confluency of 80 – 90

% in a 15 cm culture dish. One day after cytopathic effect was reached 100% of the cells, all cells were detached by pipetting up and down. Then suspension was centrifuged at 6000 x g for 5 min at 4°C. Supernatants were removed and replaced with 4 mL of fresh DMEM. The virus particles were realized from the infected cells by three cycles of freezing (5 min in liquid nitrogen) and thawing (10 min at 37°C). The lysates were clarified by centrifugation at 6.000 x g at 4°C for 10 min and stored immediately at -80°C.

Methods

For the bulk preparation of Ad, 20 x 15 cm dishes of 293A cells grown to 80 - 90 % confluency and were infected with 150 µL of the thawed seed stock. When full CPE was evident, cells were detached as before and harvested together with the supernatant. After centrifugation 6.000 x g at 4°C for 15 min, the supernatant was transferred to an extra tube and the remaining cell pellet was diluted with 3 - 4 mL of this supernatant. The rest of the supernatant was removed. Three cycles of freezing (5 min in liquid nitrogen) and thawing (10 min at 37°C) followed, then centrifugation at 6.000 x g at 4°C for 15 min was done, supernatant was transferred to a new tube to remove cell debris and kept on ice. Then 25 U Benzonase per mL lysate was used and incubated at 37°C for 30 min. Then the lysate was kept on ice again.

The CsCl purification was done by preparing a CsCl gradient in an Ultraclear Beckman tube. To do so, first 3 mL of δ=1,2 CsCl solution (13.85 g CsCl, 50 mL PBS) were added to a fresh tube and then 3 mL of δ=1,4 CsCl solution (30.5 g CsCl, 50 mL PBS) were added underneath the δ=1,2 CsCl layer w/o disturbing the interface. Then the prepared viral Supernatant was carefully added to the top of the gradient and the tube was filled up with δ=1,2 CsCl almost to the rim. Centrifugation at 32000 x g at 4°C for 1.5 h followed. After the centrifugation step three different layers should be visible within the CsCl gradient, containing cell debris in the top layer, empty virions in the second layer and in the lowest layer there should be properly packaged virions. The lowest layer was aspirated by puncturing the tube with a hollow needle into a 2 mL syringe.

For the step of desalination of the virus stock, a sephadex column was prepared by 5 x pre-washing steps with 1 x Hepes buffer. Then the virus was loaded onto the column and desalinated virus eluted in the void volume was aliquoted and stored in -80°C.

3.5.5. Endpoint dilution assay

An endpoint dilution assay was performed to determine the infectivity of the virus preparation [277]. 293A cells were used to estimate the infectivity Ad5 and its vectors and Vero cells for HSV-1. The cells were seeded in 96-well plates to a density of 10 % in 100 µL complete medium the day before infection. Next day 10-fold virus dilution series were prepared in twelve parallel wells (one dilution step for each row) in an extra 96-well plate starting with a 100-fold dilution in complete medium. 100 µL/well from each dilution between 10^-2 - 10^-9 were transferred to the cell cultures plate using the multichannel pipette. For determining virus titer of Ad and

Methods

HSV, the cytopathic effect of each virus dilution was observed for each infected well after a 1-week incubation period, whereas HCMV was analyzed after 9 days. The calculation of the TCID50/mL comprises the dilution were 50% of the cells show a cytopathic effect using the following formula:

TCID50 = A-D (S-0.5)

A - Log of highest dilution showing CPE in over 50 % of the wells D - Log of dilution factor

S - ratio of: number of wells per row with CPE versus number without CPE

For validation of the HSV resistance or sensitivity towards ACV, 10⁴ cells of the stable replicon cell line LE2D8 were seeded onto two 96-well plates and 4 h later treated in duplicates with 96, 48, 12, 6 and 1 µg/mL of ACV in 50 µL completed medium. Next the cells were infection with either the sensitive HSV-1 WT (ACV sens) or the ACV resistant HSV-1 (ACV res) at a MOI of 0.035 or mock in a volume of 50 µL. Subsequently after an incubation time of 2 days the supernatants were collected, and virus load was analyzed in duplicates by endpoint dilution assays in Vero cell line.

3.5.6. Gaussia luciferase assay

Bioluminescence measurement of Gaussia luciferase (GLuc) reporter gene expression was performed to quantify the induction of the GLuc carrying AAV replicon vectors upon virus infection. First, the GLuc assay solution was prepared according to the manufacturer’s protocol using the BioLux® Gaussia luciferase Flex Assay Kit. Aliquots of 10 mL were stored in -20°C and thawed to RT upon usage.

For the assay, supernatants were collected in different time points after induction and stored in -80°C. Right before the assay they were thawed to RT. 20 µL of each sample in technical triplicates was transferred to a white 96-well plate. Then 50 µl of the GLuc assay solution was added carefully into each well and incubated for 5 min covered for light protection. For the evaluation of GLuc expression, the emitted light was measured using an integration time of 1 second by the Tecan luminometer. For analyzing the fold induction of GLuc, the measured relative light unit (RLU) values were compared to the values measured in the supernatants of the AAV replicon vector treated but non-infected cells.

Results