Fig. 5.

The effect of a compensatory N-S194L mutation on viral virulence. (A) Location of S194L mutation in the N protein. NTD: N-terminal domain. CTD: C-terminal domain. SR: Serine/Arginine rich region. (B) Viral genomic RNA sequence analysis showing the proportion of the C28854T base (N-194L) in three mice after five passages. Three BALB/c mice were challenged with 5,000 PFU of rMA30_Nsp3-S676T. At 2 dpi, supernatants from lung homogenates from each mouse were serially passaged four more times in uninfected mice. The passaged virus was then propagated in Calu-3 cells. At 24 hpi, supernatants from infected Calu-3 cells were harvested to isolate viral RNA. Viral genomic RNA from each group was sequenced by Nanopore sequencing and compared to unpassaged rMA30_Nsp3-S676T. (C) Weight and survival of BALB/c mice infected with 5,000 PFU of rMA30, rMA30_Nsp3-S676T or rMA30_{Nsp3-S676T;N-S194L} (n = 3 for rMA30, n = 4 for rMA30_Nsp3-S676T, n = 5 for rMA30_{Nsp3-S676T; N-S194L}) (D) Viral titers and sgRNA levels from BALB/c mice after infection with 5,000 PFU rMA30 (n = 5), rMA30_Nsp3-S676T (n = 5) or rMA30_{Nsp3-S676T;N-S194L} (n = 5) infection. (E) Histopathological analysis of mouse lungs at 4 dpi after rMA30 or rMA30_{Nsp3-S676T;N-S194L} infection. Asterisks indicate pulmonary edema. (Scale bars, 170 µm.) (F) Summary of histopathological scores of lung edema (rMA30, n = 4. rMA30_{Nsp3-S676T;N-S194L}, n = 5). (G) BALB/c mice were infected with 500 PFU (sublethal dose) of rMA30 or rMA30_{Nsp3-S676T;N-S194L}. Weight and survival were recorded daily until 14 dpi (n = 5 at each group). The log-rank (Mantel–Cox) test (C and G), one-way ANOVA test with Tukey’s multiple comparisons test (D), and two-tailed, unpaired t test with Welch’s correction (F) were used to calculate P values.