Antiviral activity of Pacific oyster hemolymph against a human coronavirus – HCoV-229E


Coronaviruses can cause severe respiratory infections in humans. This study aimed to assess the antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus, HCoV-229E. An eight-fold reduction in infectivity of HCoV-229E on Huh-7 cells was observed in the presence of 10% C. gigas hemolymph.

Antiviral activity of C. gigas hemolymph positively correlated with its concentration and appears to be active during an intracellular stage of HCoV-229E infection.

Human coronaviruses are enveloped, single stranded RNA viruses that are further classified as alpha-coronaviruses (human coronavirus-229E (HCoV-229E) and HCoV-NL63) or beta-coronaviruses (HCoV-OC43, HCoV-HKU1, middle eastern respiratory syndrome (MERS-CoV), severe acute respiratory syndrome (SARS-CoV) and SARS-CoV-2) (1).

SARS-CoV-2 is a novel human coronavirus which emerged in December 2019 as the causative agent of coronavirus disease 2019 (Covid-19) (2–4). Safe and effective antiviral treatments for SARS-CoV-2 are yet to be identified, despite multiple drug repurposing attempts (5–7).

Marine molluscs represent an unexploited source of medicinal compounds (Pedler and Speck, Rev. Med. Virol, in press) (8–11). Marine molluscs lack an adaptive immune system and exclusively elicit innate immune responses (12–14), while living in an environment containing virus particles in the order of >107 per ml (15, 16).

This demonstrates the success of their strategies to prevent viral infection, which includes production of potent antiviral compounds (11, 13). In vitro inhibition of HSV-1 has been observed using extracts from the common cockle (Cerastoderma edule), greenlip abalone (Haliotis laevigata) (17), Japanese carpet shell (Ruditapes philippinarum), European flat oyster (Ostrea edulis), common whelk (Buccinum undatum) (18), blacklip abalone (Haliotis rubra) (19, 20), veined rapa whelk (Rapanosa venosa) (21) and Mediterranean mussel (Mytilus galloprovincialis) (22). Extract from the flesh of the red abalone (Haliotis rufescens) protect mice against poliovirus and influenza A (23, 24), while paolin II from the Eastern oyster (Crassostrea virginica) inhibits poliovirus (25).

Hemolymph of the Pacific oyster (Crassostrea gigas) has in vitro antiviral activity against HSV-1 and adenovirus respiratory strain 5 (AdV-5) (26–28). The major C. gigas hemolymph protein, cavortin, exerts an antiviral effect against HSV-1 after entry into Vero cells (26). Cavortin is a Mr 20,000 protein which acts as a metal chaperone (29).

Intracellular zinc has therapeutic potential for SARS-CoV-2 (30–32), and its efficacy is improved by coupling with a metal chaperone (31, 33). Crassostrea gigas has a high zinc content (34) and given cavortin’s suggested role as a metal chaperone (29), it is possible that C. gigas cavortin has potential antiviral activity against SARS-CoV-2 and may facilitate zinc transport into host cells.

The discovery of antiviral agents for SARS-CoV-2 is challenged by the limited number of laboratories with the appropriate biosafety containment level (35, 36).

HCoV-229E can be handled in lesser-rated laboratories making it more accessible for research on human coronaviruses (37) and this virus could be used for initial screening for anti-coronavirus activity. This study is the first we are aware of that assesses antiviral activity of C. gigas hemolymph against HCoV-229E.

Huh-7 cells, obtained from M. Beard were grown in Dulbecco’s modified eagle medium (DMEM) (Gibco #11965118) supplemented with 10% foetal bovine serum (FBS) (Gibco #10099141), according to standard methods (38). Twelve C. gigas oysters, grown in Coffin Bay, South Australia, were purchased from local seafood merchants. After opening, C. gigas hemolymph was extracted from the pericardial cavity using a sterile syringe and 27g needle.

Hemolymph was pooled, sterilised using a 0.2μm filter and stored at −20°C until required. Cytotoxicity of C. gigas hemolymph was determined using a trypan blue exclusion assay (26). Huh-7 cells were seeded into a 24-well plate with medium as above and 0%, 2%, 5%, 10% or 20% (v/v) C. gigas hemolymph. Cells were incubated for two days at 37°C in 5% CO2, before being stained in situ with 0.4% trypan blue (Gibco #15250061) (26).

The number of non-viable cells in three different fields of view were counted using an Olympus CK2 microscope at 40x magnification (39). The mean number of non-viable cells was lowest for 0% (1.00 ± 0.00 cells) and 2% (1.00 ± 0.47 cells) (Fig. 1). Huh-7 cell death sharply increased as hemolymph concentration exceeded 10% (Fig. 1), therefore 10% was considered an appropriate concentration for use in anti-HCoV-229E assays.

In Vero cells, C. gigas hemolymph can cause 10% cell death at a concentration of 13% (v/v) (26) or 50% cell death at 750µg ml−1 (27). The acellular and cellular fractions of C. gigas hemolymph can cause 50% cell death in Hep-2 cells at concentrations of 0.32mg ml−1 and 0.19mg ml−1 respectively (28).

FIG. 2.
FIG. 2.
non-viable (arrow) and viable (arrowhead) Huh-7 cells treated with 20% Pacific oyster (Crassostrea gigas) hemolymph.

HCoV-229E was obtained from H. Whiley. Virus titres were determined as 50% tissue culture infective doses (TCID50) (40). Huh-7 cells were seeded into 96-well plates contain either 0 or 10% C. gigas hemolymph. Three ten-fold dilutions, followed by eight two-fold dilutions were prepared using HCoV-229E stock and DMEM and inoculated into 96-well plates.

Cells were incubated at 37°C in 5% CO2 for five days before being fixed with 10% formaldehyde and stained with 0.5% crystal violet (Thermo #S25275B). Wells illustrating cytopathic effect were counted, allowing TCID50 to be calculated using the Reed-Muench method (41). When Huh-7 cells were assayed with 10% C. gigas hemolymph, an eight-fold reduction in the HCoV-229E titre (4.00 × 105 TCID50 ml−1) which is an antiviral activity of 87.5%, was observed (Table 1).

A dose-response curve was generated using 0%, 2%, 5%, 10% and 15% C. gigas hemolymph which revealed that antiviral activity positively correlated with its concentration (Table 1, Fig. 3). This is consistent with the dose-dependent antiviral activity of C. gigas hemolymph protein, cavortin, against HSV-1 (26).

Time of addition assays were used to determine the stage of HCoV-229E infection targeted by C. gigas hemolymph. In previous studies, the greatest antiviral protection of Vero cells, from HSV-1 infection was observed when C. gigas hemolymph was added 0-2 hours after infection (26, 27), suggesting that the antiviral effect was likely exerted after virus attachment and entry. An intracellular mode of antiviral action has been observed for other molluscan compounds, including lipophilic extract of H. laevigata (17) and myticin C peptides from M. galloprovincialis (22).

Here, Huh-7 cells were seeded into 24-well plates with DMEM and 10% FBS. A series of dilutions (10−4, 10−5, 10−6 and 10−7) were prepared using HCoV-229E stock and DMEM and inoculated into plates. C. gigas hemolymph was added immediately or 60 minutes after HCoV-229E. There was little difference in C. gigas hemolymph antiviral activity when it was added to Huh-7 cells immediately (98.21%) or 60 minutes after HCoV-229E infection (96.11%) (Table 2).

This suggests that C. gigas hemolymph most likely acts during an intracellular stage of HCoV-229E infection. Antiviral compounds which act during an intracellular stage of HCoV-229E infection have been identified (42–44). The macrolide and immunosuppressive compound, FK06 inhibits HCoV-229E replication in Huh-7 cells (44), while the antimalarial drug chloroquine inhibits HCoV-229E replication in epithelial lung cells (L132) by suppressing P38MAPK (43).

Thapsigargin, from the Thapsia (Thapsia garganica) plant acts during an intracellular stage of HCoV-229E infection either by inhibiting replication or activating unknown antiviral effector systems in Huh-7 cells (42).

This study reveals that C. gigas hemolymph has in vitro antiviral activity against human coronavirus HCoV-229E. This finding is relevant in the current pandemic and reinforces that C. gigas hemolymph has broad-spectrum antiviral activity. Further research is required to identify and characterise the antiviral compound(s) produced by C. gigas.

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