Figure legends:
Figure 1. (A) The location of amino acid mutations in the
three-dimensional structure of HA protein of H9N2 subtype avian
influenza virus. Yellow color indicates the locations of the HA receptor
binding sites including the positions 146-150, 109, 161, 163, 191, 198,
202, 203, and 232-237. Pink color (the positions 133, 145, 164, 168, 198
and 234) indicates mutations on antigenic sites that have been reported.
Red color (the positions 131, 181, 264, 270, 274, 278, 386 and 399)
indicates mutations that have not been previously reported as antigenic
sites. (B) HI titers of F/98 immune sera from chickens (n=8) to each
recombinant virus with single mutants in HA from the passaged viruses
occurring antigenic drift in embryonated chicken eggs or chickens. (C)
HI titers of F/98 immune sera from chickens (n=8) to each recombinant
virus with multiple mutants in HA from the passaged viruses occurred in
the 47th generation in embryonated chicken eggs under selective pressure
on antibodies. A ≥4-fold change in
HI titers of standard antiserum was considered as significant antigenic
change.
Figure 2. (A) The location of amino acid mutations in the
three-dimensional structure of HA protein of H9N2 subtype avian
influenza virus. Blue color indicates the locations of the position 198
in the HA receptor binding site. Yellow color indicates the locations of
the HA receptor binding sites including the positions 109, 161, 163,
191, 198 (blue color), 202, 203; orange color indicates the locations of
the right edge of the receptor binding pocket 232-237 and the left edge
of the receptor binding pocket 146-150. (B) MN titers of F/98 immune
sera from chickens (n=8) to the viruses F/98 or
rF/HAA198V. (C) MN titers of rF/HAA198Vimmune sera from chickens (n=8) to the viruses F/98 or
rF/HAA198V. (D) MN titers of GD/SS/94 immune sera from
chickens (n=6) to the viruses F/98, rF/HAA198V or
GD/SS/94. (E) MN titers of JS/YZ618/12 immune sera from chickens (n=6)
to the viruses F/98, rF/HAA198V or JS/YZ618/12. (F) MN
titers of rF/HAA198V immune sera from chickens (n=6) to
the viruses F/98, rF/HAA198V, GD/SS/94, or JS/YZ618/12.
A ≥4-fold change in MN titers of standard antiserum was considered as
significant antigenic change. These data indicated the fold change was
relative to the virus rF/HAA198V.
Figure 3. (A) HA mutations A198V increase receptor binding avidity of
H9N2 virus F/98, whereas S145N decreases receptor binding avidity.
Relative viral receptor binding avidities were determined by
hemagglutination of red blood cells pretreated with increasing amounts
of α2-3,6,8 neuraminidase. Data are expressed as the maximal amount of
neuraminidase that allowed full agglutination. The data are
representative of three independent experiments. (B, C) Single A198V
mutation does not affect Ab binding. Direct antibody binding to F/98 or
rF/HAA198V viruses were determined by ELISA using sera
collected from chickens vaccinated with inactivated F/98 (B) or
rF/HAA198V (C). The AUC of ELISA was calculated for
virus-Abs binding by GraphPad Prism 8 software above the value of the
corresponding negative control, which was performed under the same
conditions. Means and SD from three independent experiments. Statistical
significance was based on student’s t test (**P < 0.01;
*** P < 0.001). O.D., optical density.
Figure 4. (A) Neuraminidase activities of F/98 and
rF/HAA198V viruses, which were determined in the
fluorescence intensity of NA fluorogenic substrate. (B) Virus elution
from chicken erythrocytes. Two-fold dilutions of F/98 and
rF/HAA198V viruses were incubated with equal volumes of
chicken erythrocytes at 4 ℃ for 30 min, and the HA titers at 37℃
representing virus elution from chicken erythrocytes was monitored for 6
hours. The result was presented as the percentage of the initial HA
titer at 4℃. (C, D) Virus release assay. A198V HA mutation does not
affect viral infection (C), and prevents virus released from MDCK cells
surface significantly (D). The results were displayed as mean±SDof three independent experiments. The comparisons were performed with t
test. **Indicates very significant difference between groups
(P <0.01).
Figure 5. The cross-HI reactions between F/98 and
rF/HAA198V viruses. Three-week-old SPF chickens were
vaccinated once by subcutaneous injection of 0.3 mL of oil-emulsion of
inactivated whole virus vaccines of the F/98 and
rF/HAA198V viruses, which were inactivated by adding
0.2% formalin (v /v) for 24 h at 37 °C, respectively. At 21
d.p.v., 12 chickens from each group (the F/98 vaccine group and the
rF/HAA198V vaccine group) were bled to analyze cross-HI
titers against the F/98 and rF/HAA198V viruses.
Statistical significance was based on student’s t test (*P< 0.05).