A single amino acid change in the C-terminal domain of the matrix protein M1 of influenza B virus confers mouse adaptation and virulence

Abstract

Serial passage of an initially avirulent influenza B virus, B/Memphis/12/97, resulted in the selection of a variant which was lethal in mice. Virulence correlated with improved growth in vivo and prolonged replication. Sequencing of the complete coding regions of the parent and mouse-adapted viruses revealed 8 amino acid differences. Sequencing and characterization of intermediate passages suggested that one change in the C-terminal domain of the M1 protein, an asparagine to a serine at position 221, was responsible for acquisition of virulence and lethality. Site-directed mutagenesis of the M segment of a different virus, B/Yamanashi/166/98, to change this amino acid residue confirmed its importance by conferring improved growth and virulence in mice. This observation suggests a role for the C domain of the M1 protein in growth and virulence in a mammalian host.

Fig. 1

Growth of mouse adapted intermediates in MDCK cell culture. Viral titer was assayed at 96 hours (A) for parental virus (M0), 3^rd (M3), 6^th (M6), 9^th (M9), and 15^th (MA, mouse adapted) passages. Time course of infection (B) was assayed by measuring the HA titer of the viruses in MDCK supernatants at intervals following infection and is expressed as the log₂ reciprocal titer. No significant differences in viral growth characteristics were observed in tissue culture.

Fig. 2

Virulence of mouse adapted intermediates in mice. Groups of 6 mice were infected with parental virus (M0), 3^rd (M3), 6^th (M6), 9^th (M9), and 15^th (MA, mouse adapted) passages and followed for daily for weight loss (A) as a measure of morbidity. Mice infected with the MA virus lost significantly more weight than mice infected with other viruses. Mice infected with the MA virus also had significantly higher viral lung titers (B) both 3 days and 7 days after viral infection. An (*) indicates a statistically significant difference compared to the other groups while a double asterisk (**) indicates a significant difference compared to only the M0, M3, and M6 groups (p < 0.05).

Fig. 3

A serine at position 221 of the M1 confers mouse adaptation and virulence. Reassortant viruses containing either 7 genes from Yam98 but the M segment of Mem1297-MA, or a mutated M1 gene with the N221S change, caused more weight loss (A), high mortality (B), and higher viral titers (C) at day 3 and day 7 after infection. An (*) indicates a statistically significant difference compared to the wildtype virus rgYam98 (p < 0.05).

Fig. 4

Unique amino acids found in the C region of the M1 protein associated with mouse adaptation of influenza A and B viruses. The broad downgoing arrowhead indicates the position of the amino acid change M1 N221S reported here, while narrow upgoing arrowheads indicate the positions of changes seen in this region in other mouse adapted influenza viruses. The N and M domains are shaded. The dotted line indicates the slightly greater length of the M1 protein in influenza A viruses (252 amino acids) compared to influenza B viruses (248 amino acids). NLS = nuclear localization signal.