How to: Hepatitis D Virus Production and Infection

As with any other virus, the investigation of HDV starts with virus production. The straightforward nature of HDV’s genome makes this a relatively uncomplicated process. This protocol outlines a guide to HDV production, offering a platform to unravel the virus’s interactions with host cells. With a focus on inflammatory responses and cell-division-mediated spread, I utilized this guide to explore the intricate mechanisms underlying HDV pathogenesis.

Reagents

• HuH7 cells
• Plasmids: HDV antigenome (pJC126 — pcDNA-based eukaryotic vector), HBV envelope (pLX304- HB2.7gtB)
• DMEM
• Fetal calf serum (FCS/ FBS)
• Optional: TC supplements (1% penicillin/ streptomycin, 1% L-glutamine)
• OptiMEM
• Transfection reagent (e.g. LT-1 by Mirus #MIR2300)
• PBS
• 0.45 um strainer
• PEG8000
• Viral RNA mini kit (e.g. Qiagen #52906)
• DNase treatment kit (e.g. Promega #M6101)
• Reverse transcription kit (e.g. Thermo Fischer #362271)
• SYBR green mix (e.g. Bio-Rad #1725124)
• Forward and reverse qPCR primers targeting the HDV genome
• DMSO

All vendors are suggestions and are provided to look up manufacturer protocols.

Protocol

HDV Production:

1. Our HDV will be produced by HuH7 cells, a hepatocarcinoma cell line. These can be maintained in regular adherent cell media (DMEM, 10% FBS, 1% penicillin/ streptomycin, 1% L-glutamine). Seed the HuH7 in complete media at a density of 3.5x10⁵ /ml in 10 cm dishes. Let them adhere and grow for 24 hours.
2. Co-transfect the cells with the plasmids encoding the antigenome of HDV and the HBV envelope. Therefore mix 2.5 ug of each plasmid with 1 ml OptiMEM and 15 ul transfection reagent LT1. Of course, the DNA:cells:reagent ratio depends on the transfection reagent and is provided by the manufacturer. Incubate the transfection mix for 15 minutes at room temperature before distributing it into the media (10 ml) of the cells.
3. The day after transfection, wash cells with PBS and add fresh media.
4. Collect the supernatant at 7, 10, and 13 days post-transfection. Be careful when taking the supernatant and when adding fresh media to not disturb the producer cells. Store the supernatants at 4°C until you have collected all.
5. Once all supernatants are collected, pool and centrifuge them for 10 minutes at 500 rpm. Filtrate them additionally through a 0.45 ul strainer to remove any cells or debris.
6. Of course, these supernatants can be used directly to infect other cells. But, since they will probably contain a low titer, I suggest concentrating the virus by PEG precipitation.

PEG Precipitation:

1. Incubate your supernatants with 10% PEG at 4°C overnight.
2. Precipitate the viral particles by centrifugation at 10.000 xg for 1 h
3. Resuspend the pellet in 350 ul PBS containing 10% FBS.
4. Shake overnight at 4°C to ensure fill dissolution.
5. Centrifuge at 3000 xg for 2x 10 minutes to remove any remaining PEG.
6. Aliquot your HDV stock and store it at -20°C for short-term storage and at -80°C for long-term storage.

HDV Titration:

1. To determine the concentration of our newly produced virus stocks, you can quantify the viral RNA via RT-qPCR.
2. There are special kits to isolate viral RNA since it is fairly short and less abundant than cellular RNA. Depending on the kit, extract the RNA of 40 ul virus stock. Follow the kit's instructions.
3. Remove any remaining plasmid-derived DNA by DNase treatment. Follow the manufacturer’s instructions.
4. Because of its rod-like structure, we have to “open” the HDV genome by heat shock at 95°C for 5 minutes, followed by shock freeing at -80°C. The freezing can be performed in a metal rack stored in a -80°C freezer.
5. Measure the concentration of the carrier RNA (which is added by the viral RNA isolation kit) by Nanodrop.
6. Perform reverse transcription with 1 ug total RNA according to your kit. I used the following RT program: 25 minutes 10°C, 120 minutes 37°C, 5 minutes 85°C, and hold 4°C.
7. Dilute the cDNA 2.5x fold before qPCR.
8. To quantify the amount of cDNA, and therefore viral RNA, in our stocks, perform a qPCR with the cDNA. Mix 3 ul of your viral cDNA with 7.5 ul SYBR green mix, 3.3 ul water, 0.6 ul each, forward and reverse primers against the HDV genome. For each virus stock, work in technical triplicates. Prepare for each set of primers a standard by adding the plasmid encoding the HDV genome at final copy numbers of 10²/10⁴/10⁶/10⁸per well.
9. Perform the qPCR with the following program: 3 minutes 95°C, (40x) 10 seconds 95°C and 30 seconds 60°C, 10 seconds 95°C.
10. Along the standard, you can then
    — calculate the number of virus copies/ well,
    — take the average of the three technical replicates,
    — divide it by 3 (ul cDNA added to the qPCR),
    — multiply by 50 (ul cDNA post dilution),
    — divide by e.g. 10 (ul needed for RT),
    — and multiply by 40 (ul supernatant used for RNA extraction).
    This is your final number of copies/ ml.
11. HDV infection is performed based on international units (IU). To convert copies/ ml into IU/ ml one must extract and quantify the RNA of a reference HDV stock with a defined titer along with your new stocks:
    IU/ ml stock = (IU/ ml reference * copies/ ml stock) / copies/ ml reference

HDV Infection:

1. Seed NTCP-expressing cells (HuH7, HepaRG, A549, …) at approx. 2x10⁵ /ml in a 24-well plate 24 h before infection.
2. Infect cells with HDV at a range of 1 IU/cell in 500 ul complete (FBS + supplements) media containing 4% PEG and 1.8% DMSO per well.
3. Remove the infection medium and add fresh media containing 1.5% DMSO. Mono-infection with HDV (without HBV) ensures that no infectious progeny is produced and spread is limited to cell division. DMSO in the media will limit cell growth which is needed because of longer infection periods.
4. Cells can be stained or harvested 1–7 days post-infection.

Caution: Virus production needs approval and should be conducted in a cell culture hood of at least BSL-2.

Tips

• HepaRG cells are a human hepatic cell line commonly used in hepatitis B/D research. In contrast to HuH7, HepaRG can produce endogenous IFN which makes them an attractive cell line to study antiviral response. On the downside, they grow much slower and need special treatment in cell culture: William’s E medium, 10% HyClone FBS, 1% Pen/Strep, 1% L-glutamine, 50 uM hydrocortisone, 5 ug/ml insulin.
• To infect a cell line with HDV, it needs to express the entry receptor NTCP. Both, HuH7 and HepaRG, need to be transduced with NTCP (feel free to reach out for more information). To verify NTCP expression by fluorescence microscopy, cells can be stained with MyrcludexB-ATTO565. MyrcludexB, also known as Bulevirtide, is a peptide drug that binds to NTCP and thus inhibits HDV entry. Fuze this peptide to a fluorophore and you get a staining specific for NTCP.
• If you are interested in the cell-division-mediated spread of e.g. HDV, simply trypsinize your infected cells 6 days post-infection, and re-seed them at dilutions of 1:10–1:100 in DMSO-free complete media. Removing the DMSO will allow clonal expansion. Cells can be fixed and stained for HDAg (Hepatitis D antigen) on day 6 post-split. Caution: HepaRGs don’t like to be seeded too sparsely. Here aim for lower dilution factors. HuH7 or A549 can be seeded at higher dilution factors.