2.5 Animal experiments
In this study, 60 6-week-old BALB/c mice were obtained through Vital
River Laboratories, Beijing, China. Nasal swabs of all animals were
inoculated in 10-day-old embryonated chicken eggs and Madin-Darby-canine
kidney (MDCK) cell to confirm that mice were free of influenza viruses;
HI assays were used to confirm that animals were negative for serum
influenza virus antibody. This study was approved by the Animal Ethics
Committee of Guangxi Medical University. Animal experiments were
conducted in biosafety level 2+ containment facilities
in strict compliance with the institute’s guidelines for the care and
use of laboratory animals.
To investigate the replication and infectivity of H6N6 AIV in mice, 60
BALB/c mice were divided into 5 groups (n=12) by random number
generator. Each animal was inoculated with one strain. The mock-infected
group was inoculated with PBS. After anesthesia, each animal was
intranasally inoculated with one strain at a dose of
106 EID in 0.2 mL of PBS or with 0.2 mL of PBS. The
survial rate, weight change, temperature, and disease symptoms of
animals were recorded daily during the 14-days course of the experiment.
Animal nasal swabs were collected on days 1, 3, 5, and 7
post-inoculation and isolated and cultured in 10-day-old embryonated
chicken eggs and MDCK. Three mice in each group were euthanized on days
3, 5, and 7 post-inoculation, respectively. Tracheal and lung tissues
were collected and divided into two parts. One part was ground and
centrifuged to obtain supernatant and then isolated and cultured in
10-day-old embryonated chicken eggs and MDCK. The other part was fixed
in 10% formalin solution at room temperature for 24 hours for
pathological examination and virus protein detection. Sera were
collected on day 14 post-inoculation, and HI tests were used to detect
serum antibodies in the recovery period.