Properties of the Neuraminidase of H1N1 Viruses from the 2007–2008 Season

p-Values are the result of the Student's t test between the mean of Km, Vm, or Ki of the sensitive viruses versus either the resistant ones or the pre-2007–2008 viruses. Student's t test was also performed between the mean Km of the sensitive viruses versus the mean Km of the early seasonal virus #0006/07.

Amino acids that differ from the NC99 strain are shown in bold.

a

Except for the reference vaccine strains A/New Caledonia/20/99(H1N1) (NC99) and A/Solomon Islands//2006(H1N1) (SI06), all other viruses were A(H1N1) clinical isolates from the NIC (Northern-France) named by order number and year of isolation. Virus 0692/07 is an isolate from the 2006–2007 season.

b

Week of sampling of specimen during the 2007–2008 season.

c

IC50 determined essentially as described in [20],[30] using the MUNANA substrate at a final concentration of 100 μM.

d

The Michaelis-Menten constant Km was determined by enzymatic kinetic analyses performed on inactivated virus suspensions. Briefly, kinetics were performed using MUNANA concentrations ranging from 5 to 100 μM. Initial velocity of the reaction was calculated and plotted as a function of the MUNANA concentration. Km and Vm were calculated using a nonlinear regression of the curve according to the Michaelis-Menten equation. Vm values were expressed in arbitrary units (U/sec).

e

Hemagglutination (HA) titers were determined in duplicate by standard procedures using guinea pig red blood cells. Titers are expressed as the mean of the reciprocal of the last virus dilutions showing hemagglutination.

f

Ki determinations rely on enzymatic kinetic analyses. The kinetics were performed in the presence of variable concentrations of inhibitor (0 to 2,000 nM) and a constant MUNANA concentration (20 μM). Calculation of the Ki was performed by nonlinear regression of the plot of the initial velocity as a function of the concentration of inhibitor.

g

Km and Vm values are given as the mean of 2, 3*, 4**, or 7*** determinations.

h

Values correspond to the mean ± standard deviation of the mean Km or Vm values of the 5 pre-2007–2008 viruses , the 6 sensitive viruses, and the 11 resistant viruses. Means and p-values apply to virus groups indicated by —.

i

Ki values were determined once except for the SI06 reference strain, for which three independent determinations were performed, and the 0497/07 and 644/07 viruses, for which two independent determinations were performed.

j

Values correspond to the mean ± standard deviation of the mean Ki values of the 5 pre-2007–2008 viruses, the 6 sensitive viruses, and the 11 resistant viruses. Means and p-values apply to virus groups indicated by —.

ND, not determined; NS not significant.