Multi-Step Growth-Curve Time-Course of Human Adenovirus Type 5 in A549 Cells Quantified by Genome Copy and Infectious Titer Kinetics

A549 monolayers are synchronously infected at MOI 0.01 (multi-step) with parallel MOI 5 (single-step) reference wells, after a 1 h cold-binding / temperature shift to synchronize entry. Triplicate wells are harvested at 0, 6, 12, 24, 48, 72, and 96 h post-infection. At each time point, cell-associated and supernatant fractions are processed separately for genome qPCR and TCID50. A mock-infected control and a UV-inactivated-virus control are included. Three independent biological replicates (separate cell passages) are performed; samples coded for blinded TCID50 reading.

BSL-2; human adenovirus type 5 is a Risk Group 2 pathogen - work in a Class II biosafety cabinet with lab coat, gloves, and eye protection. Adenovirus is non-enveloped and relatively resistant to alcohol; decontaminate with 10% bleach (>=10 min contact) or an EPA-registered non-enveloped-virucidal disinfectant rather than relying on 70% ethanol alone. Bleach liquid waste to 1% final; autoclave solids. Avoid aerosols during scraping and freeze-thaw. Note replication-competent HAdV-5 (vs replication-defective vectors) requires appropriate IBC registration and personnel with no immunocompromising conditions.

Mock-infected control: defines background CPE and baseline qPCR signal (must be negative). UV-inactivated-virus control: input genomes detectable by qPCR but no rise over time and no infectious titer increase - distinguishes replication from carryover input. t=0 wash set: quantifies residual input to subtract for true replication. Single-step (MOI 5) reference: validates one-cycle kinetics for comparison. qPCR standard curve, NTC, and a positive reference DNA validate the genome assay; reference HAdV-5 stock validates the TCID50 readout.

Released infectious titer should remain near input during eclipse (~0-12 h), with cell-associated titer rising first (~24-48 h) and released titer climbing to a plateau by ~72-96 h, typically reaching 1e7-1e9 TCID50/mL for HAdV-5 in A549. Genome copies should plateau/dip early then rise 100-1000-fold during exponential phase. The genome-to-infectivity ratio is expected ~20-100:1. Mock and UV controls remain flat. Single-step MOI 5 curves show a sharper one-cycle burst.

• A549 cells (ATCC CCL-185), passage 5-25, in DMEM/F-12 + 10% FBS + 1x pen/strep
• HAdV-5 stock, single-use aliquots at -80 C
• Infection medium: DMEM/F-12, 2% FBS, 1x pen/strep
• DPBS with and without Ca2+/Mg2+
• DNA extraction kit (e.g., DNeasy Blood & Tissue) for genome qPCR
• qPCR master mix + HAdV-5 hexon primers/probe and an IVT/plasmid hexon copy-number standard
• 12-well tissue culture plates
• 0.25% trypsin-EDTA
• TCID50 reagents (HEK293 or A549 indicator cells, DMEM, crystal violet)
• Cell scrapers; freeze-thaw-compatible tubes

To generate a quantitative multi-step (low-MOI) growth curve for human adenovirus type 5 in A549 cells by harvesting cell-associated and supernatant fractions across a 0-96 h time-course and measuring both viral genome copies by qPCR (hexon gene) and infectious particles by TCID50, thereby resolving the eclipse phase, exponential replication, and release kinetics, and computing the genome-to-infectivity (particle-to-PFU) ratio.

1. Seed A549 at 2e5 cells/well in 12-well plates; grow ~24 h to ~80% confluency.
2. Chill plates and virus on ice; add HAdV-5 at MOI 0.01 (and MOI 5 references) in cold infection medium; bind 1 h at 4 C with gentle rocking to synchronize attachment.
3. Wash 2x with cold DPBS to remove unbound virus; add warm infection medium and shift to 37 C (defines t=0). Wash a t=0 set immediately to capture input.
4. At each time point (0/6/12/24/48/72/96 h), collect supernatant (clarify 2000 x g, 5 min) as the released fraction; scrape cells into 0.5 mL DPBS as the cell-associated fraction.
5. Subject cell-associated fractions to 3 freeze-thaw cycles (-80 C / 37 C) to release intracellular virus; clarify.
6. Split each fraction: one part for DNA extraction + hexon qPCR (absolute copies via standard curve), one part for TCID50 titration on indicator cells (read CPE at 7-10 days).
7. Compute copies/mL and TCID50/mL for each fraction and time point; assemble cell-associated and released growth curves.

Independent variable: time post-infection (0-96 h), with MOI (0.01 vs 5) as a secondary factor and fraction (cell-associated vs released). Dependent variables: hexon genome copies/mL and TCID50/mL per fraction, and the derived genome-to-infectivity ratio. Controlled variables: A549 passage/confluency, MOI, synchronization protocol (4 C binding then 37 C shift), harvest/processing identical across time points, freeze-thaw cycles (3x), and qPCR/TCID50 conditions.

If HAdV-5 undergoes productive multi-cycle replication in A549 cells, then after a low-MOI synchronized infection genome copies will plateau or dip during an early eclipse phase (~0-12 h), rise exponentially (~24-72 h), and reach a release plateau by ~96 h, with intracellular infectious titer rising before extracellular titer and a genome-to-infectivity ratio in the range of ~20-100:1.

Interpolate genome copies from the hexon standard curve; convert to copies/mL with dilution factors. Compute TCID50/mL by Spearman-Karber. Plot log10(copies/mL) and log10(TCID50/mL) vs time for each fraction (mean +/- SD of triplicates) in GraphPad Prism or Python/R. Subtract t=0 input where appropriate. Derive burst size = peak total virus / number of infected cells, and the particle-to-infectivity ratio = genome copies / TCID50. Phase boundaries (eclipse/exponential/plateau) are identified from the curve and the first time point of significant rise above t=0.

1. No rise in titer over time: non-synchronized or failed entry - verify 4 C binding/wash steps, confirm A549 permissiveness, use a fresh aliquot. 2. High t=0 background masking eclipse: incomplete washing - add an extra cold DPBS wash, include a wash-only control. 3. Genome rises but titer does not: defective/aggregated particles or freeze-thaw insufficient - increase to 3-4 freeze-thaw cycles, check for cytotoxicity of harvest buffer. 4. Variable TCID50 (>0.5 log10): indicator-cell confluency/passage drift - standardize indicator cells, blind-read. 5. qPCR efficiency off-range: re-prepare standard, dilute extracts to test inhibition, confirm hexon primer specificity.

Use two-way repeated-measures ANOVA (factors: time, fraction or MOI) on log10 values with Sidak/Tukey multiple-comparison correction (alpha=0.05); n=3 biological replicates with technical triplicates averaged first. Exponential-phase growth rates are compared by fitting log-linear regression and testing slope differences. Genome-to-infectivity ratios compared by t-test on log10 ratios. Power: detecting a 1 log10 difference at a given time point (SD ~0.3 log10) at 80% power, alpha=0.05, requires n=3 per group.