May 26, 2026
Preparation of Spike-in Aliquots of Murine Hepatitis Virus (MHV)
- Melissa Pitton1,
- Rachel E. McLeod1,
- Lea Carduff1,
- Ayazhan Dauletova1,
- Jolinda de Korne-Elenbaas1,
- Charles Gan1,
- Camille Hablützel1,
- Aurélie Holschneider1,
- Seju Kang1,
- Guy Loustalot1,
- Patrick Schmidhalter1,
- Linda Linda1,
- Anna Wettlauffer1,
- Daniela Yordanova1,
- Timothy R. Julian1,2,3,
- Christoph Ort1
- 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland;
- 2Swiss Tropical and Public Health Institute, Allschwil, Switzerland;
- 3University of Basel, Basel, Switzerland.
Protocol Citation: Melissa Pitton, Rachel E. McLeod, Lea Carduff, Ayazhan Dauletova, Jolinda de Korne-Elenbaas, Charles Gan, Camille Hablützel, Aurélie Holschneider, Seju Kang, Guy Loustalot, Patrick Schmidhalter, Linda Linda, Anna Wettlauffer, Daniela Yordanova, Timothy R. Julian, Christoph Ort 2026. Preparation of Spike-in Aliquots of Murine Hepatitis Virus (MHV). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5yon4l1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 11, 2026
Last Modified: May 26, 2026
Protocol Integer ID: 313005
Keywords: preparation of murine hepatitis virus, aliquots of murine hepatitis virus, murine hepatitis virus, recovery control for wastewater, total nucleic acid extraction, wastewater, wastewater yield, big batch, big batches of aliquot, preparation of spike, recovered concentration, new batch, recovery rate, quantification of the mhv stock, gene copy
Funders Acknowledgements:
Swiss National Science Foundation
Grant ID: CRSII5_205933
Swiss Federal Office of Public Health
Grant ID: 142006108/334.0-101/26
Swiss National Science Foundation
Grant ID: 142006655/334.0-107/12
Abstract
This protocol describes the preparation of Murine Hepatitis Virus (MHV) aliquots for spiking in wastewater, used as recovery control for wastewater total nucleic acid extraction. MHV is spiked in pre-extraction and the recovered concentration is measured post-extraction. This protocol also describes the quantification of the MHV stock to calculate the volume needed for spiking. Previous experiments demonstrated that spiking 400,000 gene copies per 45 mL wastewater yields a recovery rate of 5–25%. Recovery was also shown to be non-linear with the amount spiked in. Therefore, it is needed to quantify each new batch to accurately determine the volume needed to achieve the target of 400,000 gene copies per 45 mL wastewater. Big batches of aliquots are made to reduce inter-batch variability.
Materials
Consumables
- Cultivated MHV stock; aliquots of 50 µL in -80°C Freezer
- QiaAmp Viral RNA MiniKit (Qiagen: cat. no. / ID: 52904)
- 96-100% Ethanol (Sigma-Aldrich: cat. no.1.00983.101)
- PBS 10x (Thermo Fischer Scientific: cat. no. 70011044)
- Nuclease-free water (Promega: cat. no. P1195)
- 1.5 mL or 2.0 mL Eppendorf tubes (Huberlab: cat. no. 11.3829.02 / 11.3829.03)
- 1000 µL GPS LTS pipette filter tips (Mettler Toledo: cat. no. 30389274, 30389276, 30389293)
- Serological pipettes 25 mL/50mL (Huberlab: cat. no. 7.760 180/7.768 180)
- Corning‱ 250 mL PP Centrifuge Tubes with Plug Seal Cap (Corning: cat. no. 430776)
- Gloves
- Lab coat
- Permanent marker
Equipment
- Pipettes 1000 µL
- Sterile work bench
- Vortex mixer
- -80°C freezer
- Heat block at 65°C
- Benchtop centrifuge (14’000 x g)
Analytical material
This protocol uses cultivated MHV, obtained from a lab at EPFL. MHV stock is stored at -80°C until used in this protocol to prevent viral degradation.
Safety warnings
Murine Hepatitis Virus (MHV) is a beta-coronavirus, and is typically considered a Biosafety Level 2 (BSL2) pathogen that infects mice. As it is a BSL2 pathogen, working with MHV should be conducted in an appropriate laboratory in compliance with local and institutional guidelines. For example, handling MHV within a sterile bench. All material used for this protocol should be disposed of according to local and institutional guidelines, for example by sterilizing generated liquid waste and consumables before disposal.
Scope
This protocol specifically describes the preparation of the spike-in aliquots of Murine Hepatitis Virus (MHV), used during the total nucleic acid extraction from wastewater as a control to identify sample cross-contamination and estimate proportion of virus recovery through processing.
The goal of this protocol is to prepare a stock of MHV spike-in aliquots, extract the aliquots in triplicate using the QiaAmp Viral RNA MiniKit, and then estimate the amount to spike in wastewater samples.
Aliquot preparation
Clean the sterile bench with Ethanol 70% or other appropriate disinfectant and turn on heat block to 65°C.
Prepare the 1.5 mL tubes for the desired number of aliquots. Switch on the UV light for 20 minutes, if available, to disinfect surfaces.
Heat four 50 µL aliquots of cultivated MHV on a heat block until thawed.
In the meantime, prepare 100 mL 1x PBS solution, by adding 10 mL of 10x PBS to 90 mL of nuclease-free water in a 250 mL bottle (Corning).
Dilute the cultivated MHV stock 1:500 by adding the 200 µL cultivated MHV to the 100 mL 1x PBS solution.
Invent the 250 mL bottle (Corning) a few times and vortex intermittently for approximately 10-20 seconds.
Caution: Do not vortex the solution for too long as this can affect the virus.
Aliquot 510 µL into each prepared Eppendorf tube.
Dispose of the bottle (Corning).
Place the aliquots in the boxes and store at -80°C.
MHV Spike-in Aliquots quantification
Prepare the heat block at 65°C.
Take an MHV spike-in aliquot from the -80°C and thaw on the heat block at 65°C until thawed. The aliquot can be removed when only a small piece of ice remains in the aliquot to prevent overheating.
Caution: Aliquots should always be thawed on the heat block and not with hands or at room temperature, to prevent degradation of the virus due to slow thawing. Observe the sample thawing to prevent overheating, which will reduce the portion of virus.
Extract the MHV spike-in aliquot in triplicate using the QiaAmp Viral RNA Minikit (Qiagen) according to manufacturer's instructions.
Pipet 560 µL AVL buffer, 5.6 µL carrier RNA and 140 µL MHV spike-in together in a 2 mL tube. Vortex the tubes.
Incubate at room temperature (15 - 25°C) for 10 min.
Spin the tube on a table top centrifuge quickly to remove drops from the inside of the lid.
Add 560 µL ethanol (96%-100%) to each sample. Mix by vortexing.
Spin the tube on a table top centrifuge quickly to remove drops from the inside of the lid.
For each sample, place a QIAamp Mini spin column (Qiagen) in a 2 mL collection tube.
Carefully pipet 630 µL of the sample to the QIAamp Mini spin column (Qiagen).
Centrifuge at 6’000 x g (8’000 rpm) for 1 min.
Keep the spin column and discard the collection tube containing the filtrate. Place the spin column into a new collection tube.
Repeat steps 20-22.
Add 500 µL buffer AW1 (Qiagen). Centrifuge at 6’000 x g (8’000 rpm) for 1 min.
Keep the spin column and discard the collection tube containing the filtrate. Place the spin column into a new collection tube.
Add 500 µL buffer AW2 (Qiagen). Centrifuge at 20’000 x g (14’000 rpm) or max. speed of the centrifuge for 3 min.
Keep the spin column and discard the collection tube containing the filtrate. Place the spin column into a new collection tube.
Centrifuge again at 20’000 x g (14’000 rpm) or max. speed of the centrifuge for 1 min.
Place the spin column into a clean, labelled 1.5 mL plastic tube.
Open the column and add 40 µL of AVE (Qiagen) into the middle of the column.
Incubate the spin column for 2 min at room temperature and centrifuge then at 6’000 x g (8’000 rpm) for 1 min.
Repeat step 30-31, such that the total extract volume is 80 µL.
Discard the spin column.
After extraction, dilute the final extract 10x, 100x, 1000x
Quantify MHV in the dilutions in duplicate by using an appropriate assay, such as the Six-plex Digital PCR targeting SARS-CoV-2 (N1 and N2), MHV, IAV, IBV, and RSV as applied in https://dx.doi.org/10.17504/protocols.io.5jyl8xk68v2w/v.
Result processing - Calculation of spiking volume
Estimate the appropriate volume of MHV to add to each sample (in μL). In our laboratory, we targetat least 400,000 genome copy equivalents:
(400,000 genome copy equivalents / (digital PCR output in gc/μL * template volume (i.e. 5 μL) * eluate volume (80μL) / extracted volume of MHV (140μL)))
Estimate the amount of the virus to spike-in to the sample:
(rounded volume * (digital PCR output in gc/μL * template volume (i.e. 5 μL) * eluate volume (80μL) / extracted volume of MHV (140μL)))
Protocol references
Pitton, M., McLeod, R.E., Caduff, L. et al. A six-plex digital PCR assay for monitoring respiratory viruses in wastewater. Nat Water 3, 1174–1186 (2025). https://doi.org/10.1038/s44221-025-00503-x
Acknowledgements
The work published is based on Pitton et al. (2024) DOI: 10.1038/s44221-025-00503-x
This study was funded by the Swiss National Science Foundation (SNSF Sinergia grant number CRSII5_205933)
and by the Swiss Federal Office of Public Health (grant numbers 142006108/334.0-101/26 and 142006655/334.0-107/12) granted to C.O. and T.R.J.