3.3 BALB/c mice infected with H6N6 subtype AIV
To investigate the replication of the H6N6 virus in mice, we inoculated
groups of twelve 6-week-old BALB/c mice with 106 EID50
of each strain. Three mice in each group were sacrificed on day 3, 5, 7
post-inoculation, and the virus was detected in the trachea and lung.
The remaining three mice in each group were used to analyze weight
changes, disease symptoms, and death after 14 days. After inoculation of
H6N6 subtype AIV, BALB/c mice showed decreased activity, diet decline,
coarse and disordered hair, but no disease symptoms weight loss and
death. Their trachea and lung tissues were ground in homogenate and
isolated in embryonated chicken eggs and MDCK, respectively. In
embryonated chicken eggs, the virus was detected in the mice’s tracheas
and lungs inoculated with ZZ346; the HA titers on days 3, 5
post-inoculation were 1:32 and 1:16, respectively (JX20490 and ZZ1923
were not detected). Similarly, for MDCK isolation, the ZZ346 virus was
detected in the lungs of mice; the HA titers on days 3, 5
post-inoculation were 1:16 and 1:8, respectively (JX20490 and ZZ1923
were not detected).
On day 14 post-inoculation, the serum of BALB/c mice was collected, and
the HI test was used to detect the anti-influenza virus antibody in the
serum. The results showed that although ZZ1923 and JX20490 strains were
not be isolated, the serum of convalescent period was positive for the
antibody. The antibody level induced by the ZZ346 strain was higher than
that of JX20490 and ZZ1923 strains.
No obvious gross pathological changes were observed in the mice
inoculated with JX20490 and ZZ1923, while a slight hyperemia focus of 1
× 1. 2cm2 appeared in the left lung inoculated with
ZZ346 on day 5 post-inoculation. The trachea of mice inoculated with
ZZ346 and JX20490 showed different degrees of tracheal mucosal
congestion, edema, mucosal epithelial necrosis, and a small amount of
inflammatory cell infiltration. However, no obvious pathological changes
were found in the trachea of ZZ1923 inoculated mice (Figure 5 ).
In mice inoculated with ZZ346, dilatation and congestion of small blood
vessels, exudation of red blood cells, infiltration of inflammatory
cells, widening of the alveolar wall, and interlobular septum in lung
tissue were observed. Still, there were no obvious typical lesions in
mice’s lung tissue inoculated with JX20490 and ZZ1923 (Figure
5 ). Immunohistochemical detection of virus NP protein in tissue showed
that the number of cells infected by ZZ346 was more than that infected
by JX20490 and ZZ1923 (Table 3 and Figure 5 ). On days
3, 5, and 7 post-inoculation of ZZ346 strain, the NP protein of
influenza virus were detected in the trachea, bronchus, and lung tissues
of some mice. A small amount of virus NP protein could be detected in
the trachea and bronchus of mice inoculated with JX20490, but no NP
protein was detected in the lung. However, NP protein was not detected
in the trachea, bronchus, and lung tissues of mice inoculated with
ZZ1923. These results indicated that H6N6 viruses, especially the ZZ346
strain, could replicate in the respiratory system of mice without prior
adaptation.