Experiment Summary

Influenza A virus is a member of orthomyxoviridae family causing wide-spread infections in human respiratory tract. Mouse infection model is widely used in antiviral research and pathogenesis study against influenza A virus. Here, we report a protocol in infected mice with different virus doses and strains to explore how an inhibitor of lysine-specific demethylase (LSD1) impacts disease progression.

Materials and Reagents

• 1.5 ml Eppendorf tubes
• 10 µl, 200 µl, and 1 ml pipette tips
• 24-well plates
• 6-well plates
• 6 cm dish
• Syringes
• 75 cm flask (T75 flask)
• 0.22 μm filters
• Pencil
• BALB/c mice (6-8W, female)
• 293T cells
• MDCK cells
• A/WSN/33(H1N1) (WSN) virus
• A/Sichuan/1/2009 (H1N1) (SC09)
• TCP
• 1x PBS
• Isoflurane
• Picric acid (Used for labeling the mice)
• DMEM
• Paraformaldehyde
• True Blue substrate
• anti-nucleoprotein antibody
• Anti-rabbit IgG secondary antibody
• Avicel
• Bovine serum albumin (BSA)
• 2x DMEM
• Trypsin (TPCK)
• Fetal bovine serum heat inactivated (FBS)
• Agar
• 18 MΩ H₂O
• Overlay medium (DMEM + 2% FBS + 0.9% BactoTM-Agar)
• Infection medium (DMEM + 1 μg/ml TPCK)
• Trans-2-phenylcyclopropylamine hydrochloride (TCP) (1 mg/ml)
• 2.4% Avicel
• 6% BSA
• Overlay medium

Equipment

1. Pipettes
2. Dissection equipment
3. Anesthesia machine
4. Eppendorf centrifuge
5. Class II biological safety hood
6. Incubator
7. Freezer (4 °C, -20 °C and -80 °C)
8. Ultraviolet (UV) light

Procedure

A. Virus production

1. Transfection and transduction

a) Transfect monolayer of 293T cells in a 6 cm dish with 1 μg each of eight plasmids of pHW2000-WSN-segment1-8 (kindly provided by Prof. Hans Klenk, Germany) by following the manufacturer's protocol.

b) Twenty-four hours post-transfection, change the medium to 4 ml DMEM with 2% FBS, and culture at 37 °C for another 48 h.

c) Transfer all the supernatant from the transfected 293T cells to the monolayer of MDCK cells in the 6 cm dish, fill with fresh DMEM with 2% FBS if the whole volume is less than 4 ml.

d) Culture the MDCK cells for 48-96 h till CPE can be observed. Collect the MDCK supernatant and make a stock.

2. Plaque purification

a) Dilute the stock supernatant in serial 10-times dilution in PBS and apply 300 μl of each dilution onto the indicated wells of monolayer of MDCK cells plated in 6-well plate for 0.5 h.

b) After this, discard the residual medium and wash the cells in PBS for 2 times. Then add 2 ml of overlay medium (DMEM + 2% FBS + 0.9% BactoTM-Agar) on the cells and culture the cells for 2-3 days to observe plaques.

c) Encircle the plaque from beneath with a pencil and pick one plaque from the top with a 1 ml-pipette-tip, and then resuspend the plaque in 1 ml PBS at 4 °C for 16 h. This 1 ml PBS containing viruses will be used as the first-round plaque to repeat plaque purification for a second time.

d) Sequence the resulted virus and multiply in MDCK cells in a 75 cm flask to make a virus stock.

B. Virus amplification and virus titer determinations

1. Virus amplification

a) Seed monolayers of MDCK cells in a T75 flask, after 12 h cells should be 80% confluent. Then infect cells with virus at MOI = 0.001 in 15 ml infection medium (DMEM + 1 μg/ml TPCK).

b) Collect virus supernatant when cells are almost dead (about 3 days). Centrifuge at 1,000 x g for 10 min, sub-pack the supernatant and keep them in -80 °C.

2. Determine viral titer by plaque assay

a) Seed monolayers of MDCK cells in 24-well plates, after 12 h cells should be 100% confluent.

b) Then infect cells with 200 µl virus supernatants which are in ten-fold serial dilution by DMEM.

c) After a 1 h incubation at 37 °C in 5% CO₂, remove the medium and wash once with 1x PBS.

d) Then add 2 ml of overlay medium to cover the MDCK cells and incubate for 2 days, subsequently fix the cells in 4% paraformaldehyde and exposure to ultraviolet (UV) light for 30 min.

e) Perform immunostaining using an anti-nucleoprotein polyclonal primary antibody for 1 h at room temperature and an HRP-conjugated anti-rabbit IgG secondary antibody for 2 h at room temperature.

f) Finally, add 100 µl True Blue substrate to visualize the plaques.

C. The measurement of median lethal dose

1. Transfer the mice (BALB/c mice aged 6-8 weeks) to ABSL2 for adaptive feeding for 3-7 days. Divide the mice (BALB/c mice aged 6-8 weeks) randomly into four groups to infect different concentrations of influenza A virus, 4 mice for each group.
2. Dilute virus in 50 μl PBS at different concentrations (4 x 10⁴ pfu/mouse, 4 x 10³ pfu/mouse, 4 x 10² pfu/mouse, 4 x 10¹ pfu/mouse) for WSN and (9 x 10⁵ pfu/mouse, 1.3 x 10⁵ pfu/mouse, 1.3 x 10⁴ pfu/mouse, 1.3 x 10³ pfu/mouse) for SC09 (H1N1) strains.
3. Anesthetize the mice with isoflurane and infect the mice intranasally by 50 μl droplets containing different concentrations of viruses diluted in PBS.
4. Monitor the weight and death of the mice throughout the infection time course from Day 0 to Day 14 (Figure 1).
5. Calculate the median lethal dose by Statistical Product and Service Solutions (SPSS)
6. To run the probit analysis in SPSS, we follow the steps as follow: Firstly, input a minimum of three columns into the Data Editor (Number of individuals per container that responded, Total of individuals per container, Concentrations). Secondly, after columns are set, go to analyze, and press regression, then press probit. Thirdly, set your number responded column as the "Response Frequency", the total number per container as the "Total Observed", and the concentrations as the "Covariates". Don't forget to select the log base 10 to transform your concentrations. Press "OK" and then get the LD50 results.

Figure 1. Exploration of median lethal dose of WSN and SC09.

D. Mice infection with different doses of virus for different purposes

To explore the impact of TCP on mice body-weight loss and mortality, we infected mice with a lethal dose of viruses to see if treatment of TCP can rescue or worsen the disease. For this, we infected the mice with a dose of 10,000 pfu of WSN (about 5LD50) virus to guarantee a lethal infection.

1. Transfer the mice (BALB/c mice aged 6-8 weeks) to ABSL2 for adaptive feeding for 3-7 days.
2. Divide the mice (BALB/c mice aged 6-8 weeks) randomly into four groups: PBS-mock group, TCP-mock group, PBS-WSN treated group, and TCP-WSN treated group.
3. Dilute 10,000 pfu of WSN in 50 μl PBS.
4. Anesthetize mice with isoflurane and infect the mice intranasally by 50 μl droplets containing different concentrations of viruses diluted in PBS.
5. Inject 1 mg/ml TCP (100 μl/mouse) and PBS (100 μl/mouse) intraperitoneally once a day for 10 days.
6. Monitor the weight and death of the mice throughout the infection time course from Day 0 to Day 14.

After we know that TCP accelerate the disease, we then decide to infect the mice with a sublethal dose of virus, 500 pfu of WSN virus (0.25LD50) (Figure 2) or 300 pfu of SC09 (0.25LD50), wherein the impact of TCP can be observed more clearly (Figures 4C and 4D of Shan et al., 2017). For sublethal infection of WSN virus, all the groups of animals could survive till at least Day 9 post-infection (Figure 2), so that infected tissues can be collected for histological analyses. The procedure is almost as above except that the mice are sacrificed at Day 9 for further analysis.

Fig. 2 Low doses of viruses were used to guarantee mice of all groups could survive till at least Day 9 post-infection.