Viral Growth Kinetics and Quantification

Growth curve analysis was performed as described previously (Ma et al., 2018 (link)). Briefly, the growth kinetics of US1 mutant viruses were compared to that of the parental strain. Cell cultures were infected at an MOI of 0.01 (multi-step assay) or 2 (single-step assay). After 2 h of adsorption, the cells were washed and then overlaid with DMEM containing 2% NBS. Supernatants and infected cells were separately harvested at 24, 48, 72, 96 h (multi-step assay) or 6, 12, 18, 24, 36 h (single-step assay) at successive intervals, and the amount of infectious virus was determined by plaque assay using DEF cells.
The viral dosage and sampling time points were the same as for the growth curve detection described above. First, the UL30 DNA fragment was amplified with primer P8, and the fragment diluted in a 10-fold gradient was used as a template followed by the addition of primer P9 and probe P10 for qPCR to construct a standard curve: Y = −4.262X+43.675. Primers P8, 9, and 10 used in this part are listed in Table 1. The viral DNA in the samples was extracted using a Viral RNA/DNA Extraction Kit (Takara, China). qPCR was performed after the addition of primer P9 and probe P10, and viral DNA copies were estimated at various time points using the standard curve. All of the analyses were performed independently in triplicate with the standard error.