Animal experiment ethical statement
Twelve female specific-pathogen-free (SPF) BALB/c mice, aged 6–8 weeks old, were purchased from the Experimental Animal Center of Zhejiang Province, China. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang Province. The study was approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine, (Ethical approval No. 2017.402-1). All experiments with H7N9 virus were performed in a bio-safety level 3 laboratory of the First Affiliated Hospital, Zhejiang University School of Medicine (Registration No. CNAS BL0022).
Cells and viruses
The Madin-Darby canine kidney cell line (MDCK) was obtained from the ATCC (Rockville, MD, USA). The cell line was propagated in growth medium comprising Dulbecco's modified Eagle's medium (DMEM; Cat#11965092, Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Cat#10100147, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2. The A/Guangdong/GZ8H002/2017(H7N9) virus used in this study was isolated from a patient in Guangzhou, China, in 2017 (GenBank: MF455313-455320). Virus stocks were propagated in the allantoic cavities of 9-day-old specific pathogen free (SPF) embryonated chicken eggs at 37 °C for 72 h. The allantoic fluid was harvested and tested using a haemagglutinin (HA) assay, in which 50 μl of allantoic fluid solution was double diluted at 1:2 with phosphate-buffered saline (PBS; Cat#20012500BT, Gibco, Grand Island, NY, USA) in a 96-well blood coagulation plate. Then, an equal volume of 1% chicken red blood cells were added, and observed at room temperature for 30–45 min. The highest dilution at which blood coagulation appears is the HA titre. Aliquots of the allantoic fluid containing the virus were stored at −80 °C until further use.
Determination of the virus TCID50
MDCK cells were inoculated into 96-well cell culture plates at 3 × 104 cells/well (in 100 μl). The virus was diluted in viral growth liquid 10 times continuously, from 10-1 to 10-10, and the last two rows were reserved for control. After cells grew into a single layer in the 96-well plate, the medium was discarded, the wells were washed once with sterile PBS, and the diluted virus (100 μl/well) was added to the wells, with each concentration infecting cells in four wells. A normal cell control well (virus free) was set. The 96-well plate was incubated at plate at 37 °C in a 5% CO2 incubator for 2 h, and then washed two times with PBS. The normal control hole and the virus infection wells then received virus growth fluid (DMEM with 1% cow serum albumin; 100 μl/well). The plate was incubated at 37 °C in a 5% CO2 incubator for 72 h. The HA assay was then used to identify positive or negative wells. The median tissue culture infectious dose (TCID50) was calculated as we did in the previous study [25].
Virus inoculation
Mice were inoculated intranasally with 50 µl 106 TCID50 A/Guangdong/GZ8H002/2017(H7N9) virus. The same volume of PBS was given to the mice in the control group. Mice were observed for signs of illness, weight loss, and death post-infection. Then, at 2, 3, and 7 days post-infection (dpi), some of the mice were sacrificed. Their serum and organs (lung, brain, heart, kidney, liver, and spleen.) were collected. Part of the organ was fixed in 10% buffered formalin, while the other part was used to isolate the virus and detect virus levels using quantitative PCR.
The histopathology of organ tissue
Organ tissue was prepared for haematoxylin eosin (HE) staining. Immunohistochemical (IHC) assays were also conducted. Paraffin sections of organs were de-waxed and then subjected to heat treatment in citrate buffer. Endogenous peroxidase activity was quenched using 0.3% H2O2 in methanol. Sections were blocked for 2 h with 3% bovine serum albumin (BSA; Cat#H1130, Solarbio, Tongzhou, Beijing, China) in PBS and incubated sequentially overnight at 4 °C with 1:200 dilution of polyclonal rabbit anti‑H7N9 antibodies (Cat#GTX125989, GeneTex, Irvine, CA, USA) for 12 h. Antibody binding was detected using EnVision System reagents (Cat#K5007, DAKO, Glostrup, Denmark). All slides were counterstained with haematoxylin and eosin.
Isolation of the virus from mouse serum and organs
The serum samples collected at 2, 3, and 7 dpi were used to isolate the virus. About 100 µl of serum was injected into the allantoic cavities of 9-day-old SPF embryonated chicken eggs. The embryonated chicken eggs were cultured in an incubator at 37 °C for 72 h. The allantoic fluid was harvested and tested using an HA assay.
The tissue leachate was obtained as follows: One ml of sterile PBS was added to the frozen tissues in storage tubes, and then the tissue was cut using sterile scissors in the biosafety cabinet. The tubes were centrifuged at 500 g for 10 minutes. Then, 200 μl of the supernatant was used to isolate the virus. The virus separation of tissue leachate refers to the serum virus separation procedure.
Determination of the virus titre of organs
One ml of sterile PBS was added to the frozen tissues in storage tubes, and then the tissue samples were cut using sterile scissors in the biosafety cabinet. The tubes were centrifuged at 500 g for 10 minutes. Then, 200 μl of the supernatant was added with 800 μl Trizol to extract RNA. The amount of virus was then evaluated using quantitative real-time PCR. A H7N9 nucleic acid quantitative detection kit (Cat#Z-RR-0309-02) purchased from Zhijiang biological technology Co., Ltd. (Shanghai, China) was then applied. The steps were as follows: 19 μl of H7N9 nucleic acid PCR detection reaction mixture and 1 μl quantitative PCR enzyme were mixed by vortexing and then centrifuged at 500 g for several seconds. This mixture was added to the PCR reaction tube, together with 5 μl of the RNA sample, for a total reaction volume of 25 μl. The tube was covered, centrifuged briefly, and subjected to the following PCR conditions: 40 cycles of 45 °C for 10 minutes, 95 °C for 15 seconds, and 60 °C for 60 seconds. The relative quantity of H7N9 virus was determined by the cycle threshold (Ct) value.
Statistical analysis
Statistical analyses of the weight data and the survival rate were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).