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. 2022 Jan 3;5(1):5.
doi: 10.3390/mps5010005.

Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells

Tautvydas Shuipys  1 , Naim Montazeri  1
Affiliations

Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells

Tautvydas Shuipys et al. Methods Protoc. .

Abstract

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Keywords: CCL-1; CCL-9.1; COVID-19; MHV; SARS; cell passage; coronavirus; plaque assay; surrogate.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Scheme 1
Scheme 1
Visual representation of the complete workflow from cell revival to MHV propagation and quantification. The time required for each step is listed in parenthesis. The workflow begins with cell maintenance including (1) revival of frozen cells. Afterwards, (2) the cell lines are passaged until stable cell counts and viability are obtained. At this point, (3) preparation of freeze stocks is recommended before starting (4) MHV propagation, (5) MHV harvesting, and (6) conducting a plaque assay with (7) staining to quantify infectious viral titer.
Figure 1
Figure 1
Typical appearance and confluency of cells prior to cell passage. (a) NCTC clone 1469; cell culture medium was removed prior to this photo (see Section 3.2.1 step 22). (b) NCTC clone 929. Scale bar represents 100 µm.
Figure 2
Figure 2
Cytopathic effect (CPE) of MHV infected NCTC clone 1469 cells after 19 h. Infected cells form syncytium: a multinucleated mass formed by the fusion of cells. Scale bar represents 1 mm.
Figure 3
Figure 3
Example of MHV plaque assay wells in titration of two separate virus samples. (a) Solid agarose overlay; 4 h after staining with Neutral Red; infected with MHV (5.49 log PFU/mL) at a 10−3 dilution. (b) Methyl cellulose overlay; 4 h after fixing/staining with crystal violet in formaldehyde; infected with MHV (6.59 log PFU/mL) at a 10−4 dilution. The different dilutions of MHV shown in (a,b) were to present a similar countable range of plaques across the two virus samples. Scale bar represents 5 mm.
See this image and copyright information in PMC

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