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.