Sequencing-based Neutralization Assay for Influenza A Virus

Thaw sera On ice

Prepare RDE solution, by adding 20mL into one receptor destroying enzyme (RDE) bottle (VWR, Cat. No. 370013). Replace stopper and gently invert to mix. Sterile filter the solution through a 0.22 um filter prior to use. Aliquots of this RDE solution can be stored at -20 C for future use, if needed.

Pipette RDE solution into labeled, externally threaded tubes, such that you can dilute the sera 1:4. For our sera samples, we added 300µL . Then, added 100µL.

To removes residual sialic acids in serum incubate serum and RDE at 37°C 2h 30m 0s

Then, to heat-inactivate the RDE and serum, incubate at 56°C 0h 30m 0s

Move samples to ice, and then store heat-inactivated and RDE-treated serum samples at -80°C until you are ready to begin the neutralization assays.

Different plate setups may be used depending on how many samples of sera you are testing, and how many dilutions you wish to run for each serum. For example, you could perform serial dilutions of serum down the plate vertically, running up to 12 serum samples with 7 different dilutions (plus a no-serum control) per serum sample. Or you could perform dilutions across the plate horizontally, running 8 serum samples with 11 different dilutions per serum-sample (plus a no-serum control). Additional plate set up designs are also feasible to meet the needs of your particular project.

Pictured below is an example plate setup for 12 serum samples with 7 concentrations of serum

Thaw virus and RDE-treated sera Room temperature 0h 30m 0s

Pick an initial serum dilution factor for your assay: this will be the highest serum concentration tested, and other wells will contain serial dilutions of this concentration. You also need to choose what dilution factor to use as you dilute the serum from one well to the next. For example, you might start with an initial dilution of 1:20 and perform 3-fold dilutions down the plate. Recall that the RDE treated sera has already been diluted 1:4 in the steps above.

Add influenza growth media (Opti-MEM supplemented with 0.1% heat-inactivated FBS, 0.3% bovine serum albumin, 100 µg per mL of calcium chloride, 100 U per mL penicillin, and 100 µg

per mL streptomycin) to your plate such that all rows contain either 50 uL or the volume needed for your initial dilution of sera.

An example is shown below, using the setup described above where 8 serum samples are run horizontally across the plate. In this example, we use an initial dilution of serum of 1:20. As the serum has been diluted 1:4 during RDE treatment and will be diluted 1:2 with virus in a later step, you'd need to dilute the RDE-treated sera 2:5 for the initial dilution in the plate to obtain a final dilution of 1:20.

For this example, you would add 45µL to the wells in your initial dilution column and 50µL in every other well:

Add 30µL to each well in column 2. This leaves you with a 1:10 sera dilution of the original serum (a 2:5 dilution of RDE-treated serum that was already diluted 1:4 during RDE treatment) in 75uL total volume in the wells in column 2.

Perform serial 3-fold dilutions, pipetting 25µL from column 2 into column 3, and mixing by pipetting up and down several times. Repeat this step with column 3 into column 4 and so forth until you get to row 12, after which you will pipette the residual 25 uL from this column directly into bleach. After this step, you will have completed the serial 3-fold dilutions across the plate. You should still have no serum in column 1, and all 96 wells should now contain 50uL total volume.

The appropriate amount of virus library needed for each plate is experimentally determined as the amount of virus that can be added to the number of cells per well (50,000 MDCK-SIAT1 cells as described below) and still be in the linear range of viral transcriptional output changing with viral dilution. (See note for more detail). As 50µL of virus will be added to each well, we first dilute the virus library that the appropriate amount of virus is in each 50µL aliquot, and then add 50µL to each well (including the control, no serum row of wells).

Incubate plate of virus and serum 37°C 1h 0m 0s

After the virus-serum mix has been incubated for 35 min of the 60 min incubation time, prepare cells to be added to the plate. First, aspirate media off of a confluent 100 mm plate of MDCK-SIAT1 cells. Wash the cells once with 2mL. Then, treat cells with 2mL , incubating at 37°C until cells disassociate from the plate (this typically takes around 5-10 min, since MDCK-SIAT1 cells are highly adherent). Once the cells are mostly dissociated, pipetting gently can help dissociate the remainder.

Inactivate the trypsin by adding 4mL , washing the cells off the plate and resuspending in this media. Transfer the resuspended cells to a conical tube. Centrifuge 300x g , then aspirate the supernatant. Wash cells once with 5mL , and then resuspend cells in 5mL . Use a cell counter to determine cell concentration, and dilute cells to 1e6 cells/mL with more influenza growth media. You will need about 5.5 mL of cells at 1e6 cells/ml in influenza growth media for each 96-well plate that you wish to run.

Once the plate with the serum and the virus library has been incubating for 1 hour total, add 50µL to each well.

Return the plate containing cells, serum, and virus library to the 5% CO2 incubator. Incubate at 37°C for 16h 0m 0s. This time allows non-neutralized viruses to infect cells and transcribe viral RNA.

Approximately 15 hours into the incubation period of the cells and virus, start thawing reagents from the iScript Select Synthesis Kit (BioRad) needed to perform cDNA synthesis (5x reaction mix, nuclease-free water, GSP enhancer, gene-specific primer, iScript enzyme mix) and a 200 pM aliquot of the RNA spike-in control. Most of the reaction mix components can be thawed Room temperature but the the enzyme mix and the RNA spike-in control should be thawed On ice.

When your RNA spike-in control is thawed, mix 55µL into 5445µL, thereby diluting the spike-in control 1:100 for a final concentration of 2 pM. You will end up with 5.5 mL total volume of lysis buffer using these specific amounts, which is sufficient for one plate. Adjust the above amounts according to how many plates of neutralization assays you set up.

When your cDNA synthesis reagents have thawed, prepare a cDNA synthesis mastermix. We use an 18 µL final reaction volume for each well, typically preparing sufficient mastermix for 100 reactions per plate to allow excess prior to distributing the the mastermix to PCR tubes. For one plate, your mastermix can be prepared like so:

400µL

900µL

200µL

200µL

100µL

Please adjust these amounts according to how many plates of neutralization assays you are running.

Aliquot 18µL into 8-strip PCR tubes or a 96-well PCR plate.

Remove the supernatant from the plate using a multichannel pipette, taking care not to disturb the cells and pipette this directly into bleach solution.

Wash the infected cells once with 150µL per well. This wash step removes any residual virus from the supernatant and improves the cell lysis efficiency. Take care not to disturb the adhered cells when performing this wash step. When removing this liquid, pipette directly into bleach, and rinse tips with bleach solution. Examine the wells of the plate to ensure that residual liquid is completely removed from the wells of the plate prior to adding lysis buffer. This is essential as if residual liquid remains, this will dilute the spike-in control and result in noise in the measurements collected with this assay.

Add 50µL (with the RNA spike-in control added) to each well. Wait 2-5 minutes for cells to lyse. Examine wells under the microscope to determine that cell lysis has occurred. This lysis buffer is gentle, expect that nuclei will remain adhered to the plate, though the cell borders will be less defined following lysis.

After 2-5 minutes, transfer lysate to a new 96-well PCR plate so that the lysis of cells will not continue.

Add 2µL to the strip tubes (or 96-well PCR plate) containing 18µL

Transfer the strip tubes (or 96-well PCR plate) containing cDNA synthesis reaction mixes to the thermocycler with the lid heated to 60°C

Set the following protocol:

Incubate at 42°C 1h 0m 0s

Inactivate enzymes at 85°C 0h 5m 0s

Hold at 4°C

*This is a potential stopping point, samples could be stored a -20°C overnight, if needed before progressing to the next step of the assay.

During cDNA synthesis, prepare Round 1 PCR mixes.

For one sample, the mix is as follows:

1.5µL

1.5µL

25µL

17µL

5µL

= 50µL

The following mix is enough to prepare for 96 samples:

150µL

150µL

2500µL

1700µL

=4500µL

Aliquot 45µL per well in a 96-well PCR plate.

Add 5µL . Seal the 96-well PCR plate with a PCR plate seal that is appropriate for thermal cycling and cold storage, such as ( #MSF1001, BioRad ).

Set the PCR plate containing Round 1 PCR reactions in thermocycler with the lid heated to 100°C

Set the following protocol:

Initial denaturation

95°C 0h 2m 0s

Denaturation

95°C 0h 0m 20s

Annealing

56°C 0h 0m 10s

Extension

70°C 0h 0m 20s

Repeat previous three PCR cycle steps for denaturation, annealing, and extension 19 more times

Final extension

70°C 0h 2m 0s

Hold 10°C

*This is a potential stopping point, samples could be stored a -20°C overnight, if needed before progressing to the next step of the assay.

Prepare mix for Round 2 PCR. For one sample, the reaction mix is as follows:

2.4µL

20µL

16.6µL

1µL

= 40µL

We prepare a mastermix of KOD and H2O and apply this first to the plate, then add the primers. The following mix is enough to prepare for 96 samples:

2000µL

1660µL

= 3660µL

Transfer 36.6µL into each well of a 96-well PCR plate.

Add 2.4µL (each mix of primers should match the number assigned to your sample).

Add 1µL .

Seal the 96-well PCR plate with a PCR plate seal that is appropriate for thermal cycling and cold storage.

Set 96-well PCR plate containing the Round 2 reaction mixes in thermocycler with the lid heated to 100°C

Set the following protocol:

Initial denaturation

95°C 0h 2m 0s

Denaturation

95°C 0h 0m 20s

Annealing

66°C 0h 0m 10s

Extension

70°C 0h 0m 20s

Repeat previous three PCR cycle steps for denaturation, annealing, and extension 19 more times

Final extension

70°C 0h 2m 0s

Hold 10°C

*This is a potential stopping point, samples could be stored a -20°C overnight, if needed before progressing to the next step of the assay.

Prepare a 1% agarose gel. This can be done during Round 2 PCR.

Pool Round 2 PCR products at equal volume (enough so you have at least ~200uL total volume).

*We pool using a multichannel, first pooling all columns of the plate into a single 8-strip (5 uL from each sample), then pooling all 8 of the pools for each row of the plate. This will result in total volume of 480 uL per plate. It is not necessary to prepare this total volume for sequencing, only approximately ~100 uL needs to be gel extracted and bead purified for sequencing, but we have found it useful to pipette a larger volume (5 uL as opposed to 2 uL) per sample when pooling to ensure that all samples are included in the final pool.

Run 100µL on your gel from step 33 at 85 V 0h 40m 0s . We often run 30-40µL of the sample per well on the agarose gel (with loading buffer), this results in 3-4 lanes of sample to be cut out and processed for sequencing.

Cut the band out of the gel, and extract the DNA using a Nucleospin Gel Extraction Kit (740609, Takara), or alternate gel extraction kit. Transfer the eluate to a strip tube for magnetic bead cleanup with AMPure XP SPRI Reagent (Beckman Coulter).

Perform a magnetic bead cleanup on your sample using AMPure XP SPRI Reagent to remove residual salts from the gel extraction:

Add 2X the volume of your sample of AMPure XP SPRI Reagent (i.e. if you eluted in 40 uL add 80 uL Ampure XP beads).

Incubate Room temperature 0h 5m 0s .

Load strip of tubes onto magnetic block. Incubate 0h 5m 0s .

Pull off liquid while being careful to avoid disrupting magnetic beads. Wash twice, using 150µL each time (again, taking care to not disrupt the magnetic beads). Extract all liquid from tube and wait for pellet to start drying.

When the pellet is dry (but not so dry that it has started to crack, approximately 0h 10m 0s ) resuspend in 40µL . Remove strip of tubes from magnetic block and repeatedly pipette the liquid up and down to thoroughly mix the beads into the elution buffer. Incubate atRoom temperature for 0h 5m 0s .

Place strip tubes on magnetic block, incubate 0h 5m 0s .

Once the beads are adhered to the magnet, remove the residual liquid from the beads, careful not to disturb the beads, and transfer this liquid to new 1.5 uL tube.

Quantify total DNA concentration, for this we use a Qubit. We typically obtain 40µL of sample at 0.005µg/µL

Label, appropriately dilute, and submit sample for sequencing as determined by the sequencing service you are using. We use Illumina NextSeq for these runs (a P1 run for 1 plate and a P2 should be used for 2-4 plates). We submit the 181 bp amplicon for sequencing with a 50 bp read length. We aim to obtain an average coverage of between 500,000-1,000,000 reads/well. This is likely higher coverage than necessary, however, by over-sequencing we allow for variability in loading of the different wells, which is helpful given that we are not performing any sample normalization prior to pooling.

Following demultiplexing of the sequencing run, sequencing data is analyzed using the modular analysis pipeline developed by the Bloom lab for processing high-throughput sequencing-based neutralization assays. This pipeline is available at https://github.com/jbloomlab/seqneut-pipeline

See associated manuscript for a detailed description of this analysis method.

Briefly, this pipeline takes in the FASTQ files from a sequencing run, calculates the counts for each barcoded viral variant and the RNA spike-in control in each well. The ratio of barcoded viral variant to spike-in control in each well containing serum is normalized to the ratio of barcoded viral variant to spike-in control in the no-serum control well to calculate a fraction infectivity. Then, neutralization titers are computed by fitting Hill-curve style neutralization curves to the fraction infectivity values using the neutcurve package; see the documentation for the details of these curves. The titers represent the reciprocal serum dilutions at which half the viral infectivity is neutralized. For more details, please see the description at https://github.com/jbloomlab/seqneut-pipeline