2.6 Replication of H6N6 virus in human respiratory tissuein vitro
This study was approved by the Medical Ethics Committee of Guangxi Medical University. Five lung tissue cases, which were collected during surgery, were provided by the First Affiliated Hospital of Guangxi Medical University. The selected specimens had no related respiratory tract infectious diseases. The specimens were sent to the laboratory immediately upon collection, and the suspected cancer tissues and/or other tissues with lesions were removed. The normal bronchial and lung tissues were cut into 0.2×0.2×0.2cm3. Two blocks of bronchial and lung tissues from humans were selected to detect whether the samples were infected by influenza viruses. One of the blocks was grinded to isolate and culture the virus; the other one was used to detect viral antigen by immunohistochemistry and to ensure that the specimens used for the assay were free from influenza virus infection.
The bronchial and lung tissues were placed into a 6-well cell culture plate, repeatedly rinsed with F-12K tissue culture medium containing antibiotics and L-glutamate, inoculated with 106TCID50 of the virus in a volume of 500μL medium, and then cultured at 37°C and 5% CO2 for 1h. 500μL of PBS was added into one well and used as a mock control. The tissue blocks were then rinsed with F-12K culture medium containing 0.2% TPCK-trypsin and 1%BSA and further incubated with the above medium. Two tissue blocks were collected at 12h, 24h, and 48h post-inoculation of the virus, respectively, where one was ground in cold PBS and homogenated, and then the supernatant was collected and inoculated into 10-day-old embryonated chicken eggs and MDCK cells. TCID50 was used to determine the virus titer. The other one was fixed in 10% formalin for 24 hours for pathological examination and detection of viral protein.