tetano
Editor, Senior Moderator
J Virol Methods
. 2021 Oct 8;114317.
doi: 10.1016/j.jviromet.2021.114317. Online ahead of print.
Improving human coronavirus OC43 (HCoV-OC43) research comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2
Erin E Schirtzinger[SUP] 1 [/SUP], Yunjeong Kim[SUP] 1 [/SUP], A Sally Davis[SUP] 2 [/SUP]
Affiliations
Abstract
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 dose[SUB]50[/SUB](TCID[SUB]50[/SUB])/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 TCID[SUB]50[/SUB] 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 ˜10[SUP]7[/SUP] 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 ˜10[SUP]6.5[/SUP] TCID[SUB]50[/SUB]/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 TCID[SUB]50[/SUB] 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.
Keywords: HCoV-OC43; SARS-CoV-2; coronavirus; flow virometry; virus titration.
. 2021 Oct 8;114317.
doi: 10.1016/j.jviromet.2021.114317. Online ahead of print.
Improving human coronavirus OC43 (HCoV-OC43) research comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2
Erin E Schirtzinger[SUP] 1 [/SUP], Yunjeong Kim[SUP] 1 [/SUP], A Sally Davis[SUP] 2 [/SUP]
Affiliations
- PMID: 34634321
- DOI: 10.1016/j.jviromet.2021.114317
Abstract
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 dose[SUB]50[/SUB](TCID[SUB]50[/SUB])/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 TCID[SUB]50[/SUB] 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 ˜10[SUP]7[/SUP] 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 ˜10[SUP]6.5[/SUP] TCID[SUB]50[/SUB]/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 TCID[SUB]50[/SUB] 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.
Keywords: HCoV-OC43; SARS-CoV-2; coronavirus; flow virometry; virus titration.