Animal experiment ethical statement
Specific-pathogen-free (SPF) female BALB/c mice (n=31, 6–8 weeks old) were obtained from the Experimental Animal Center of Zhejiang Province, China. All animal experiments were carried out following the principles of the Guide for the Care and Use of Laboratory Animals of Zhejiang Province. The Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine, approved the present study. A bio-safety level 3 laboratory at the First Affiliated Hospital, Zhejiang University School of Medicine was used to perform all the H7N9 virus experiments (Registration No. CNAS BL0022).
Viruses and cells
The American type culture collection (Rockville, MD, USA) provided the Madin-Darby canine kidney cell line (MDCK). MDCK cells were cultured using Dulbecco's modified Eagle's medium (DMEM; Cat#11965092, Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; Cat#10100147, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a 5% CO2 atmosphere. The present study used a A/Guangdong/GZ8H002/2017(H7N9) virus (GenBank: MF455313-455320) isolated from an infected patient in Guangzhou, China, in 2017. Viruses were grown in the allantoic cavities of 9-day-old SPF embryonated chicken eggs at for 72 h 37 °C. The harvested allantoic fluid was tested using a haemagglutinin (HA) assay. Fifty microliters of allantoic fluid was diluted at 1:2 using phosphate-buffered saline (PBS; Cat#20012500BT, Gibco) 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 appeared was the HA titre. Allantoic fluid aliquots containing the virus were placed in a −80 °C freezer until further use.
Virus median tissue culture infectious dose (TCID50) determination
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 the controls. After cells grew into a single layer in the 96-well plate, the medium was removed, the wells were rinsed with sterile PBS once, 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 37 °C in a 5% CO2 incubator for 2 h, and then washed two times with PBS. The normal control well and the virus infection wells then received virus growth fluid (DMEM with 1% FBS; 100 μl/well). The microtitre plate was incubated for 72 h in a 5% CO2 incubator at 37 °C. The HA assay was then used to identify positive or negative wells. The TCID50 was calculated according to a previously published method [25].
Inoculation of the virus
Fifty microliters of 106 TCID50 of A/Guangdong/GZ8H002/2017(H7N9) virus was inoculated into the mice intranasally. The control group (n=5) received the same volume of PBS. The mice (n=14) were observed for signs of death, weight loss, and illness post-infection. Some mice (n=12) were sacrificed humanly at 2, 3, and 7 days post infection (dpi). Their serum and organs (spleen, liver, kidney, heart, brain, and lungs) were excised or collected. The organs were dived into parts, one of which was fixed in 10% buffered formalin, and the other subjected to viral isolation and quantitative PCR to detect virus levels.
Organ histopathology
Haematoxylin eosin (HE) staining of organ tissues was performed. The organ tissues were also subjected to Immunohistochemistry (IHC) staining. Organ tissues were embedded in paraffin and sectioned. The sections were then de-waxed and heated in citrate buffer. H2O2 (0.3%) in methanol was used to quench endogenous peroxidase activity. Sections were blocked for 2 h with Three percent bovine serum albumin (BSA; Cat#H1130, Solarbio, Tongzhou, Beijing, China) in PBS was used to block the sections for 2 h. The sections were then incubated at 4 °C overnight (12 h) with a 1:200 dilution of rabbit polyclonal antibodies recognizing H7N9 (Cat#GTX125989, GeneTex, Irvine, CA, USA). EnVision System reagents (Cat#K5007, DAKO, Glostrup, Denmark) were used to detect the bound antibodies. HE was used to counterstain all the slides.
Virus isolation from mouse serum and organs
Virus isolation was attempted from serum sampled at 2, 3, and 7 dpi. The allantoic cavities of 9-day-old SPF embryonated chicken eggs were injected with approximately 100 µl of serum and cultured in an incubator for 72 h at 37 °C. Thereafter, the allantoic fluid was sampled and subjected to an HA assay.
The tissue leachate was obtained as follows: 1 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 from the tissue leachate was similar to serum virus separation procedure.
Determination of the virus titre of organs
One millilitre 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 then centrifuged at 500 ´ g for 10 minutes. Then, 200 μl of the supernatant was added with 800 μl Trizol to extract the RNA. Quantitative real-time PCR (qPCR) was then used to calculate the amount of the virus. qPCR was carried out using an H7N9 nucleic acid quantitative detection kit (Cat#Z-RR-0309-02; Zhijiang biological technology Co., Ltd. (Shanghai, China). Nineteen microlitres of H7N9 nucleic acid PCR detection reaction mixture and 1 μl of 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
GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used for the statistical analyses of the survival rate and weight data. Statistical analyses of the data for weight was conducted using Mann-Whitney test. P-values< 0.05 were considered statistically significant.