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.