Experimental Adaptation of Murine Norovirus to Calcium Hydroxide

FIGURE 1. Schematic depiction of serial passages as performed in experimental adaptation of murine norovirus (MNV). Once an initial infection is completed in plate 1, the virus is titrated and the desired amount is used to infect the cells in plate 2. The process was repeated five times.

FIGURE 2. Time-series inactivation rate of murine norovirus obtained in serial passages without (blue) and with (red) lime treatment in the first (A) and second (B) rounds. Each point represents an average value from three replicates.

FIGURE 3. Inactivation rate of murine norovirus (log reduction value: LRV) obtained in serial passages without (−lime) and with (+lime) lime treatment 5 min after exposure to lime solution. Each point represents an average value from three replicates. Solid lines are linear regression lines of best fit. Asterisks indicate a statistically difference between the −lime lineages and +lime lineages (p < 0.01 by Mann–Whitney U test).

FIGURE 4. Time series of nucleotide diversity at non-synonymous sites (piN) and synonymous sites (piS) in each protein coding gene [open reading frames (ORFs) 1, 2, and 3] in the first (A) and second (B) rounds. Each passage time is described as the number on panels. Solid lines represent the 1:1 relationship between piS and piN indicative of natural selection and neutral evolution.

FIGURE 5. Trajectories of single-nucleotide polymorphisms (SNPs) detected with at least 1% frequency through the serial passage with (+lime) and without (−lime) treatment in the first (A) and second (B) rounds.

FIGURE 6. Log reduction values (LRVs) of infectious particles produced using a plasmid-based reverse genetics system [clone MNV (A6089G)] and the laboratory strain used as the founder in the serial passages with lime treatment (ancestor MNV). Asterisks indicate a statistically difference between the two viruses (p < 0.01 by Mann–Whitney U test).