Determination of the capsid breakpoint temperature.

The breakpoint temperature, or melting temperature, of the viral capsids was determined by a thermal-shift assay. The assay was performed in an PCR thermocycler as described above, but each PCR tube was held at a different temperature ranging from 25 to 70°C at 2° intervals and was incubated for 1 min at each temperature. The breakpoint was identified as the temperature at which the inactivation rate shifted from low to high. It was determined by fitting a segmental linear regression to a plot of ln(N/N₀) versus temperature applied, where N/N₀ represents the residual fraction of infectious virus titers after the 1-min inactivation period (Fig. 8). The breakpoint temperature, reflected by the intersection of the two linear regression lines, were determined together with the respective standard error. Each breakpoint was derived based on at least two pooled experimental replicates.

Determination of the breakpoint temperature. The figure shows the examples of CVB5-Faulkner and CVB5-L061815 at a NaCl concentration of 0.01 M. The ln of the residual infectivity (N/N₀) after 1 min of treatment is plotted as a function of treatment temperature. The data are fitted to a segmental linear regression (black lines). The intersection of the two linear portions defines the breakpoint temperature of each virus.