key: cord-0771162-62p7n1f4
authors: Hayashi, Kyoko; Asai, Satomi; Umezawa, Kazuo; Kakizoe, Hidehumi; Miyachi, Hayato; Morita, Masanobu; Akaike, Takaaki; Kuno, Hitoshi; Komatsu, Satoko; Watanabe, Takumi; Kawahara, Toshio
title: Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS‐CoV‐2 as assessed by a plaque assay
date: 2021-11-27
journal: J Clin Lab Anal
DOI: 10.1002/jcla.24146
sha: e6781709c6dc3ae33fceba24904f88872f48fe9a
doc_id: 771162
cord_uid: 62p7n1f4

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of coronavirus disease 2019 (COVID‐19) and is capable of human‐to‐human transmission and rapid global spread. Thus, the establishment of high‐quality viral detection and quantification methods, and the development of anti‐SARS‐CoV‐2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS‐CoV‐2 particles using a plaque assay with 0.5% agarose‐ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36–40 h post‐infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS‐CoV‐2 in a time‐ and concentration‐dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS‐CoV‐2.

Due to a new emerging RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1 social and economic activities worldwide have come to a complete standstill. The virus has been identified as a coronavirus that is closely related to the 2003 SARS-CoV. 2 The rapid spread of SARS-CoV-2 has taken a lot of scientific effort for developing diagnostic, antiviral, and vaccine countermeasures. Therefore, there is an urgent need for the establishment of a high-quality viral detection and quantification method and the development of anti-SARS-CoV-2 agents. SARS-CoV-2 is a single-stranded positive-sense RNA virus whose genome encodes four structural proteins, the nucleocapsid protein N that is a component of the capsid, the membrane protein and the envelope protein that are involved in the virus budding process, and the spike glycoprotein that is a key player in binding cell receptor and membrane fusion and virus entry into host cells. 3 In general, RNA viruses have higher mutation rates than DNA viruses, 4-6 because most RNA viruses have single-stranded RNA and weak ability to repair gene mutations, while most DNA viruses have double-stranded DNA and high ability to repair the mutations. These mutations may sometimes weaken the efficacy of vaccines and/or antiviral agents that inhibit viral proliferation. That is, the purpose of the vaccine was production of the antibody against the viral proteins that bind to cells. When mutations occur in the viral proteins, however, the antibody might lose its binding ability to the mutant viruses, resulting in weakened vaccine efficacy. In comparison, virucidal agents that destroy viruses might be less susceptible to the mutations in viral RNA. Thus, an ideal antiviral drug should exhibit broad-spectrum activity against many variants, for example, by destroying the structure of envelope and/or capsid. Therefore, the plaque assay remains the gold standard in determining the numbers of infectious virions. 10 Monogalactosyl diacylglyceride (MGDG) is one of glycoglycerolipids contained in such as vegetables, fruits, and grains. 11 MGDGs from algae have been also reported as the components with anti-tumor and anti-inflammatory activities. 12, 13 Previously, we reported the virucidal activity of MGDG obtained from a microalga, Coccomyxa sp. KJ, against herpes simplex virus type 2 (HSV-2), 14 an enveloped virus that causes genital herpes. 15, 16 Physical changes in HSV-2 shape including a decrease in virus particle size and possible damage to the viral envelope were observed after MGDG treatment, as assessed using electron microscopy. In accordance with possible damage to the envelope, MGDG-treated HSV-2 showed no pathogenicity in an animal model.

As SARS-CoV-2 is also enveloped virus, MGDG might cause any damage to the envelope, resulting in the loss of binding ability to host cells.

In this study, we describe the rapid quantification of infectious SARS-CoV-2 using a plaque assay and the evaluation of virucidal activity of MGDG against the virus by plaque titration. Additionally, we established conditions for the determination of plaque number within two days post-infection and also confirmed the inactivation of the clinical isolates of SARS-CoV-2 by MGDG.

MGDG was obtained as an anti-HSV component from Coccomyxa sp. KJ, and its chemical structure has been determined to consist of α-linolenic acid (C18:3) (approximately 72%) and 7, 10, 13-hexadecatrienoic acid (C16:3) (approximately 23%) of the total fatty acids as reported previously. 14 

The VeroE6/TMPRSS2 cell line, expressing transmembrane protease 

The plaque assay was performed on VeroE6/TMPRSS2 cells. The cells (1 × 10 6 cells/dish) were grown in 35-mm dishes and infected with 10fold serial dilutions of the virus at room temperature (26°C) for 1 h.

Subsequently, 2 ml of an overlay medium was added. The overlay medium contained 1% SeaPlaque™ agarose (Lonza, Rockland, ME, USA), 0.8%, 1.2%, or 2% methylcellulose (MC) (4000cP, Wako Pure Chemical Industries, Ltd.), or 0.5% agarose-ME (Medium Electroosmosis) (classic type, Nacalai Tesque, Inc., Kyoto, Japan) in 1% FBS-DMEM. At 1, 2, or 3 days post-infection, the cells were fixed with 10% formaldehyde in phosphate-buffered saline (PBS) for 2 h at room temperature.

The overlay medium was then removed, and the cell monolayers were stained with 0.06% crystal violet solution (20% ethanol in distilled water). The staining solution was removed to reveal the plaques.

Virucidal assays have been used to determine whether a compound inactivates free viral particles outside of cells. The assay is performed F I G U R E 1 Dendrogram of SARS-CoV-2 strains obtained from clinical specimens by incubating the virus with the test compound for a specified time followed by the determination of the remaining infectious viral particles.

In the present study, MGDG was tested at three concentrations of 10, 100, and 1000 μg/ml against four isolates of SARS-CoV-2 [1 × 10 5 plaque-forming units (PFU)/ml]. The remaining virus infectivity was calculated using a plaque assay as follows: MGDG and the virus samples were mixed and incubated at room temperature for 10, 30, 60, 180, and 360 min. PBS was tested in parallel as the negative control. Following the incubation period, the solutions were diluted 100-fold with PBS and each dilution was added to the cell monolayer in 35-mm dishes at 100 μl/dish. The cells were incubated at room temperature for 1 h, overlaid with DMEM containing 0.5% agarose-ME, and further incubated at 37°C in a 5% CO 2 incubator.

After 36-40 h incubation, the dishes were observed for plaques.

Several overlay media were evaluated for plaque formation in virusinfected cells. Small plaques were formed after incubation for two to three days with 0.8% MC-DMEM overlay medium, while smaller plaques were formed with 1.2%-2% MC-DMEM medium. These plaques did not form a clean circle. Large plaques were formed after incubation for two days with 1% SeaPlaque-DMEM overlay medium; however, the rate of plaque formation was so fast that it was sometimes difficult to determine the appropriate fixation time. However, clear circular plaques of appropriate size appeared after 36-40 h post-infection in the cell cultures incubated with 0.5% agarose-ME-DMEM overlay medium. Therefore, 0.5% agarose-ME-DMEM overlay medium was used in the subsequent virucidal assays.

Whole RNA sequences of the clinical isolates were compared with the sequence of reference strain Wuhan-Hu-1. The sequence of clinical isolate #1 was much similar to the sequence of reference strain than those of clinical isolates #2, #3, and #4 ( Figure 1) . Interestingly, clinical isolates #2, #3, and #4 have a mutation D614G in the spike protein became dominant early in the COVID-19 pandemic and this mutation increases entry efficiency with enhanced ACE2-binding affinity. 18 

We assessed the virucidal effect of MGDG on SARS-CoV-2. The assay was based on incubating the MGDG-virus mixture prior to titration of the remaining virus infectivity via a plaque assay. 

There are many reports on the methods used to measure infectious SARS-CoV-2 particles by plaque assays with 0.375%-1% agarose, 0.6% or 1.2% microcrystalline cellulose, 1% MC, 2% Noble agar, 3% carboxymethylcellulose, or 0.8% Avicel RC-581 in the overlay medium (Table 1) . [19] [20] [21] [22] [23] [24] [25] [26] [27] In these plaque assay conditions, two to four days are required to detect the plaques. On the contrary, in this study, we have described an assay that allows faster quantification of SARS-CoV-2 by detecting plaques formed in the cell monolayers within 36-40 h post-infection using 0.5% agarose-ME overlay medium.

There was no marked difference in the in vitro susceptibility of the four clinical isolates of SARS-CoV-2 to MGDG, regardless of mutation in the spike protein that affects binding activity to ACE2 ( Figure 3 ). It is important to elucidate the virucidal mechanism of MGDG against SARS-CoV-2. As shown in a previous report, 14 In conclusion, we have developed a rapid method for quantifying SARS-CoV-2 using a plaque assay. Additionally, we have described the inactivation of the virus by MGDG isolated from a microalga, Coccomyxa sp. KJ, using agarose-ME overlay medium.

In summary, we expect that the method of the plaque assay reported here will help determine the neutralizing antibody titers in serum samples such as those of COVID-19 patients, where the loss of viral infectivity by serum can be determined using a plaque assay.

We also expect that MGDG could effectively act as a novel anti-SARS-CoV-2 agent through direct (virucidal) pathways.

Transfer Program through Target-driven R & D (A-STEP) Grant

Number JPMJTR204H. We would like to thank Editage (www.edita ge.com) for English language editing.

The authors declare no competing interests. 

The data that support the findings of this study are available from the corresponding author, K.H., upon reasonable request.

https://orcid.org/0000-0002-9582-1201

Hayato Miyachi https://orcid.org/0000-0002-5343-6751

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

A new coronavirus associated with human respiratory disease in China

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines

The spike protein of SARS-CoV -a target for vaccine and therapeutic development

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1 (Supplementary Appendix)

Assessment of plaque assay methods for alphaviruses

Oral administration of monogalactosyl diacylglycerol from spinach inhibits colon tumor growth in mice

Anti-tumourpromoting glyceroglycolipids from the green alga, Chlorella vulgaris

Antiinflammatory potential of monogalactosyl diacylglycerol and a monoacylglycerol from the edible brown seaweed Fucus spiralis Linnaeus

In vitro and in vivo anti-herpes simplex virus activity of monogalactosyl diacylglyceride from Coccomyxa sp. KJ (IPOD FERM BP-22254), a green microalga

Epidemiology of herpes simplex virus type 2 infection in the developing world

Genital herpes: A Review

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus

Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

SARScoronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Propagation, inactivation, and safety testing of SARS-CoV-2

An optimized highthroughput immuno-plaque assay for SARS-CoV-2. Front Microbiol

Growth, detection, quantification, and inactivation of SARS-CoV-2

A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: a bedside-to-bench study

Two detailed plaque assay protocols for the quantification of infectious SARS-CoV-2

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay