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.