Cell culture
Madin–Darby canine kidney cells (MDCK), human embryonic kidney cells (293T), human type II alveolar epithelial cells (A549), chicken embryo fibroblasts cells (DF-1) and porcine kidney cells (PK15) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), HEPES (10 mM; Invitrogen), penicillin (100 units/ml), and streptomycin (100μg/ml; Invitrogen), and were incubated in a humidified atmosphere of 5% CO2 at 37°C.
Site-directed mutagenesis
The eight gene plasmids of HuN virus were constructed and stored by the Chinese National Influenza Center. A plasmid with a signal mutation A271T in the PB2 protein was constructed using the forward primer 5’-GTAAGAAGAGCAACAGTGTCAGCAG-3’ and the reverse primer 5’-CTGCTGACACTGTTGCTCTTCTTAC-3’. To ensure the presence of the point mutation and the absence of unwanted mutations, the viral cDNA was sequenced.
Virus rescue and virus titration
As previously described [23], the eight plasmids containing the cDNA of the HuN271A (with residue 271A in PB2 protein) or HuN271T (with residue 271T in PB2 protein) virus were co-transfected into co-cultured 293T/MDCK cells on six-well plates using SuperFect transfection reagent (Qiagen, Valencia, CA, USA). After 48 hours, cultured supernatants were inoculated into 9-day-old specific pathogen free (SPF) embryonated eggs. To avoid unwanted mutations, we sequenced both the recombinant viruses. Virus titration was performed in MDCK cells. The 50% tissue culture infective dose (TCID50) was calculated by the Reed-Muench formula [24].
Virus growth kinetics study
To detect the growth kinetics of both recombinant viruses, A549, DF-1 and PK15 cells were inoculated with the viruses at the multiplicity of infection (MOI) of 0.01. After incubating at 37°C for 1 hour, the supernatants were removed and cells were washed twice with PBS. DMEM containing TPCK-trypsin, penicillin and streptomycin was added. Supernatants were collected at specified time points of 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours post inoculation (hpi) and stored at -80 °C until use. Virus titers were determined by a TCID50 assay in MDCK cells.
Animal experiments
To evaluate the contribution of the substitution PB2-T271A to pathogenicity of the EA H1N1 SIVs, we compared the infectivity, replication and virulence of recombinant viruses in mice. Eight-to ten-week-old SPF C57BL/6J female mice were used in the experiment. Mice were randomly assigned. Each group had five mice. The mice were anesthetized with pentobarbital sodium and inoculated intranasally with 50 μl of 10-fold serial dilutions of each recombinant virus in phosphate-buffered saline (PBS), with doses ranging from 101-106 TCID50. Mock mice were inoculated with 50 μl PBS. Body weight were recorded daily for 14 days post inoculation (dpi). When the mice lost 30% of their body weight, they were killed humanely. The 50% mouse lethal dose (MLD50) was calculated based on the survival rate of the mice. Serum from the surviving mice was collected on the 14th day after post-infection, and we detected hemagglutinin inhibiting (HI) antibody titers to calculate the 50% mouse infective dose (MID50).
To evaluate the tropism and replication capacity of both recombinant viruses, we inoculated mice with 105 TCID50 viruses. Three mice were euthanized at 1, 4, and 7 dpi, respectively. Respiratory tract samples containing lung, trachea, and nasal turbinate were collected. The right lobes of the lungs were pumped with 10% formaldehyde solution, which were used for histological analysis. The viral titers were determined by TCID50 assay in MDCK cells.
Histopathological analysis
The right lungs were fixed and dehydrated. The tissue blocks were placed in a mold containing melted paraffin wax and solidified into wax blocks. Lung tissues were sectioned at 4–5 μm thick. Then hematoxylin and eosin staining (H&E) was performed to observe viral infection.
Quantification of cytokines in mice lungs
The severity of influenza virus in human and mice was closely related to the expression of cytokines and chemokines, including interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), chemokine (C-C motif) ligand 5 (CCL5), and interferon α1 (IFN-α1) [25-29]. RNA was extracted from the mice lungs (QIAamp Viral RNA Mini Kit). Levels of cytokine and chemokine proteins, including IL-6, IL-8, IL-10, CCL5, and IFN-α1 were detected by quantitative PCR. Quantitative PCR was conducted by the reagent of TB Green Premix Ex Taq (TaKaRa Biotech, Dalian, China). The LightCycler 96 was used to detect the amplification (Roche, USA).
Polymerase activity assay
Monolayers of 293T cells were transfected with a firefly luciferase reporter plasmid, a Renilla luciferase expressing plasmid and four plasmids expressing the polymerase genes PB2, PB1, PA and NP. The Renilla luciferase was used as an internal transfection control for the dual luciferase assay. After 24 hours of transfection, luciferase activity was measured using EnVision 2104 Multilabel Reader (PerkinElmer, USA) and the assay was performed in triplicate.
Western blot assay
293T cells transfected with plasmids in the polymerase activity assay were lysed with passive lysis buffer (Promega, Madison, USA). Cells lysates were mixed with SDS-PAGE sample loading buffer (Beyotime Biotech, China) and heated at 95 ℃ for 10 min. The samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, USA). The membrane was blocked with 5% nonfat milk for 2 hours and then incubated with anti-PB2 (GTX125926, GeneTex, Irvine, USA). The primary antibody was incubated overnight at 4 ℃. The membrane was incubated with goat anti-rabbit secondary antibody for 1 hour. The images were obtained by using the MiniChemi Imaging and Analysis System (Sage Creation, China).
Statistical analysis
The student’s t test and two-way ANOVA were calculated using GraphPad Prism software 6.0. A P value < 0.05 was considered to be statistically significant.