2 Methods
2.1 DDVax Production Process
Summary
2.1.1 Uninfected Cell
Culture
Serum-Free Vero cells (2 x 104cells/cm2) were grown in OptiPro Serum-Free Media
(SFM) (Lifetech, A31343) with Glutamax (Gibco, 35050-061) at 37°C and
5% CO2. Cells were expanded into 3 x 10-Layer
CellStacks (6360 cm2) (Corning, 3271) and 1 x 1-Layer
CellStacks (636 cm2) (Corning, 3268). For each
passage, cells were seeded at either 2.0 x 104cells/cm2 for 48 ± 8-hours or 1.5 x
104 cell/cm2 for 72 ± 8-hours. Cell
harvest was performed using TrypLE Select (Life Tech, 12563092). Cells
were centrifuged at 500 x g for 5 minutes at 18°C and resuspended in
OptiPro SFM. Cell enumeration was performed using a Vi-Cell Cell
Viability Analyzer.
2.1.2 Generation of DDVax Pilot
Material
Upon achieving cell confluency within the 3 x 10-Layer or 1 x 1-Layer
CellStacks, Vero cells were infected with the DDVax Research Virus Stock
(RVS), Lot # N16-5-20-RV at a multiplicity of infection (MOI) of 0.0005
PFU/cell. Infection volume used for each 10-Layer CellStack was 1300ml
and the infection volume for the 1-Layer CellStack was 130ml. Infected
cultures were then incubated at 37°C and 5% CO2 for
72-hours. Following the 72-hour infection incubation, flasks were
examined for cytopathic effects (CPE). DDVax virus was then harvested by
pumping the supernatant from each CellStack into a 5L Flexboy bag. The
harvested pool underwent Benzonase treatment to digest Host Cell DNA
(HCD). A 500mM MgCl2 solution was added to the Flexboy
bag to achieve a final concentration of 1.5mM MgCl2 for
Benzonase treatment. Benzonase (EMD Millipore, 101679) was added at a
concentration of 50,000 U/L. The Flexboy bag was thoroughly rocked to
mix and then incubated for 60 minutes at 37°C and 5%
CO2, with rocking at 10-minute intervals. The
Benzonase-treated pool was divided into 500ml conical tubes and
centrifuged at 3000 x g for 15 minutes at 18°C to remove large cell
debris. Supernatant containing the DDVax, now the clarified pool, was
pumped out of the centrifuge bottles into a new 5L Flexboy bag. Virus
was then concentrated by a factor of 6 using ultrafiltration (UF) using
the KMPi TFF system (Repligen) over a 500kD Hollow Fiber Membrane
(Repligen, S02-E500-05-N, 500kD, 20cm length, 0.5mm fiber diameter).
Concentration was performed at a target shear rate of 3000
s-1 and TMP setpoint of 5 psi. The concentrated UF
pool was diafiltered (DF) into a buffer containing 0.2M NaCl, 10mM
Sodium Phosphate, 4% Sucrose, 5mM Glutamax, pH 7.4 ± 0.1. Buffer
exchange was performed for 10 diavolumes. Diafiltration was performed at
a target shear rate of 3000 s-1
2.2 DDVax sequencing and
analysis
Illumina shotgun sequencing libraries were prepared from total RNA using
the Kapa RNA HyperPrep kit following the manufacturer’s protocol. Dual
indexed libraries were sequenced on an Illumina NextSeq 500 sequencer to
generate single-end 150 nt reads.
We used two complementary approaches to detect and quantify viral
variants. First, we used the lofreq tool to identify single nucleotide
variants and short insertions and deletions (Wilm et al., 2012). Second,
we used DI-tector to identify structural variants including longer
deletions and insertions and copy back defective viral genomes
(DVGs)(Beauclair et al., 2018; Vignuzzi & Lopez, 2019). These tools
were run as part of a reproducible Nextflow pipeline, available at
https://github.com/stenglein-lab/viral_variant_caller/releases/tag/DDVax_paper_release.
Software dependencies and reference sequences (DDVax) are captured in
this version-controlled release and in the conda environment contained
therein.
To quantify variants, adapter-derived and low-quality bases were trimmed
using Cutadapt(Martin, 2011). Host cell-derived reads were removed using
bowtie2 to align reads to the Chlorocebus sabeus genome,
accession GCF_000409795.2(Langmead & Salzberg, 2012). Host- and
quality-filtered reads were aligned to the S, M, and L segment
RVFV/DDVax reference sequences using the BWA aligner(Langmead &
Salzberg, 2012; Li & Durbin, 2009). The reference sequences consisted
of the RVFV-derived portions of the DDVax plasmid sequences. To improve
accuracy of structural variant (indel) calls, base quality scores were
recalibrated using GATK (McKenna et al., 2010). Single nucleotide
variants (SNVs) and structural variants were called using LoFreq(Wilm et
al., 2012). The minimum depth of coverage to call a variant was set at
40x coverage. SnpEff and SnpSift were used to predict the functional
impact of variants(Cingolani, Patel, et al., 2012; Cingolani, Platts, et
al., 2012). Variant calling distinguished between variants that were not
detected despite sufficient data and positions that lacked sufficient
data to call variants. Defective viral genomes were identified using the
DI-tector tool(Beauclair et al., 2018). Outputs of these analyses were
tabulated, processed, and visualized in R. Variants with frequencies ≥
3% were reported(Grubaugh et al., 2019).
2.3 Virus strains
Stocks of DDVax were produced as described above. The passage history of
MP-12 strain is unknown, but DQ375404.1 (L segment), DQ380208.1 (M
segment) and DQ380154.1 (S segment) sequences were confirmed by Sanger
sequencing. ZH501 strain virus was obtained from R. Bowen. V1 (Vero)
passage stock was passaged twice in Vero cells to obtain V3 stocks used
for this study.