Improved plaque assay for human coronaviruses 229E and OC43

Bracci, Nicole; Pan, Han-Chi; Lehman, Caitlin W.; Kehn-Hall, Kylene; Lin, Shih‐Chao

doi:10.7717/peerj.10639

Cited by 25 publications

(38 citation statements)

References 30 publications

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“…In contrast, HCoV-OC43 produced CPE (cell lysis, cell rounding and vacuolization) in MRC-5 cells starting on day 2 which continued through day 7. Also, contrary to some previous reports (Bracci et al 2020; Hirose et al 2021), CPE was observed in HCoV-OC43 infected Vero E6 cells. Cell rounding and lysis began on day 3 and continued until day 7 (Fig.…”

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confidence: 99%

“…“the common cold,” and are typically restricted to the upper respiratory tract. Also, a review of extant literature finds researchers using a variety of methods to quantify HCoV-OC43 employing different cell lines, HRT-18 (human colon cancer cells) (Lambert et al, 2008), RD (human rhabdosarcoma cells) (Schmidt, Cooney, and Kenny, 1979), MRC-5 (human lung fibroblasts) (Collins, 1993; Collins, 1995; Kim et al, 2019), BSC-1(African green monkey kidney cells) (Bruckova et al, 1970), Vero E6 expressing TMPRSS2 (Hirose et al, 2021) and Mv1Lu (mink lung cells) (Bracci et al, 2020), using different titration assay types (either TCID 50 or plaque assay), the latter with different types (agarose, Avicel, methylcellulose) and percentages of overlays, employing different incubation temperatures (33°C or 37°C), times (2-7 days) and detection methods (immunoperoxidase with chromagen, immunofluorescence, neutral red, crystal violet). This lack of standardization hampers evaluation and comparison of study results, as the different cell lines may have different growth kinetics and susceptibilities to virus grown in heterologous cells.…”

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“…3a-c) that quickly grew into each other at lower dilutions during the four-day incubation. Bracci et al (2020) tested HCoV-OC43 in a plaque assay using MRC-5 cells with a five-day incubation at 37°C. They concluded that plaques were countable, but that MRC-5 cells were too slow-growing and variable to further optimize the assay (Bracci et al 2020).…”

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confidence: 99%

“…Bracci et al (2020) tested HCoV-OC43 in a plaque assay using MRC-5 cells with a five-day incubation at 37°C. They concluded that plaques were countable, but that MRC-5 cells were too slow-growing and variable to further optimize the assay (Bracci et al 2020). The incubation time for plaques to develop in MRC-5 cells may need to be shortened to less than four days to prevent the plaques from growing together or a higher percentage overlay used such that its viscosity better restricts the movement of the virus.…”

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Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2

Schirtzinger¹,

Kim²,

Davis³

2021

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ~107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ~106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

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Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2

Schirtzinger¹,

Kim²,

Davis³

2021

Preprint

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“…TCID 50 values were calculated by the Spearman & Kärber algorithm as previously described. 48 OC43 plaque assay was carried out on RD cells as described by Bracci et al ( 49 )…”

Section: Methodsmentioning

confidence: 99%

Metal-Mediated Nanoscale Cerium Oxide Inactivates Human Coronavirus and Rhinovirus by Surface Disruption

et al. 2021

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The COVID19 pandemic has brought global attention to the threat of emerging viruses and to antiviral therapies, in general. In particular, the high transmissibility and infectivity of respiratory viruses have been brought to the general public’s attention, along with the need for highly effective antiviral and disinfectant materials/products. This study has developed two distinct silver-modified formulations of redox-active nanoscale cerium oxide (AgCNP1 and AgCNP2). The formulations show specific antiviral activities toward tested OC43 coronavirus and RV14 rhinovirus pathogens, with materials characterization demonstrating a chemically stable character for silver nanophases on ceria particles and significant differences in Ce 3+ /Ce 4+ redox state ratio (25.8 and 53.7% Ce 3+ for AgCNP1 & 2, respectively). In situ electrochemical studies further highlight differences in formulation-specific viral inactivation and suggest specific modes of action. Altogether, the results from this study support the utility of AgCNP formulations as high stability, high efficacy materials for use against clinically relevant virus species.

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Improved Culture Methods for Human Coronaviruses HCoV‐OC43, HCoV‐229E, and HCoV‐NL63

Fausto,

Otter,

Bracci

et al. 2023

Current Protocols

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HCoV‐OC43, HCoV‐229E, HCoV‐NL63, and HCoV‐HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, these four so‐called seasonal HCoVs generally cause mild upper‐respiratory‐tract illness. As Biosafety Level 2 (BSL‐2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high‐titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV‐OC43, ‐229E, and ‐NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV‐OC43, ‐229E, and ‐NL63 in order to generate high titers (Vero E6, Huh‐7, and LLC‐MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Growth of HCoVsBasic Protocol 2: Quantification of HCoV by plaque assayBasic Protocol 3: Quantification of HCoV RNA products of replicationBasic Protocol 4: Concentrating HCoVs via ultracentrifugation

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Improved plaque assay for human coronaviruses 229E and OC43

Cited by 25 publications

References 30 publications

Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2

Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2

Metal-Mediated Nanoscale Cerium Oxide Inactivates Human Coronavirus and Rhinovirus by Surface Disruption

Improved Culture Methods for Human Coronaviruses HCoV‐OC43, HCoV‐229E, and HCoV‐NL63

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