Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses

ABSTRACT

Little is known about the antiviral response in mollusks. As in other invertebrates, the interferon signaling pathways have not been identified, and in fact, there is a debate about whether invertebrates possess antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naive mussels confer resistance in oysters to ostreid herpesvirus 1 (OsHV-1) when oyster hemocytes are incubated with mussel hemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel hemolymph and hemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel hemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible for the antiviral activity of mussel hemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks. Moreover, myticin C peptides showed antiviral activity against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications.

IMPORTANCE Several bioactive molecules that have potential pharmaceutical or industrial applications have been identified and isolated from marine invertebrates. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel hemolymph and hemocytes, showed potential as an antiviral agent against ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster-farming sector. Both hemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster hemocytes. Additionally, a modified peptide derived from myticin C or the nanoencapsulated normal peptide also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of mollusks.

INTRODUCTION

Adaptive immunity, which is a characteristic of vertebrates, is based on the recognition of “nonself” structures by the major histocompatibility complex (MHC), T cell receptors, and antibodies, as well as other effector molecules. However, the innate immune system is the only response present in invertebrates, and it is able to recognize conserved pathogen-associated molecular patterns (PAMPs), common structures present in different microorganisms (1, 2), and even danger signals (3, 4). Furthermore, this “primitive” nonspecific immune system has contributed to the survival of these animals and their evolution for more than 450 million years (5–7), which suggests that it must provide some biological advantages.

Aquatic animals in general, and bivalve mollusks in particular, are continuously exposed to different stress conditions due to the permanent and close contact with their natural environment, especially when feeding: bivalves filter large amounts of water, and this activity results in permanent exposure to microorganisms, which can be concentrated in their bodies. The level of contact between the environment and bivalves is so close that these animals are also known as sentinels in toxicology because of the major role they play as biomarkers of contamination (8).

Since 2008, massive mortality associated with ostreid herpesvirus 1 (OsHV-1) outbreaks has been reported in Europe (mainly in France) among Crassostrea gigas spat and juveniles (9–15). Later, the virus was associated with major mortalities in Pacific oysters from Australia and Asia (16–18). Today, herpesviruses are considered an important threat to the worldwide production of Pacific oysters.

The family Herpesviridae comprises enveloped viruses with a large, linear, double-stranded DNA genome that cause several diseases in animals, including humans. In particular, herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are major human pathogens responsible for long-term latent infections, with periods of recurring viral replication (19). Due to the lack of effective vaccines, the moderate to high toxicity of the available antiherpes compounds, and the appearance of resistant viral strains, new inhibitors for these viruses have been extensively researched (20, 21).

The aims of this work were to confirm that mussels (Mytilus galloprovincialis) are more resistant than oysters to the ostreid herpesvirus 1 microvariant (OsHV-1 μVar), one of the most important bivalve pathogens, and to determine the putative role of myticin C, a constitutively expressed molecule in mussels, in resistance against herpesviruses.

MATERIALS AND METHODS

Animals.

Adult M. galloprovincialis mussels and C. gigas oysters, 8 to 10 cm in shell length, were purchased from a commercial shellfish farm (Vigo, Galicia, Spain) and maintained in open-circuit filtered seawater tanks at 15°C with aeration. The animals were fed daily with Phaeodactylum tricornutum and Isochrysis galbana. Prior to the experiments, the animals were acclimatized to aquarium conditions for at least 1 week.

Viruses and cells.

The OsHV-1 μVar was kindly provided by Tristan Renault (IFREMER, France) as a viral suspension obtained from infected oyster hemocytes (22). An HSV-1 clinical isolate (>99% homology to the isolate SK087 US4-6 genes; GenBank accession number AY240815.1) and an HSV-2 clinical isolate (>99% homology to the isolate 99-62039 US4 gene; GenBank accession number HM011430.1) were provided by the Virology Service of Hospital Universitario Central de Asturias (Oviedo, Spain), propagated in Vero cells (European Collection of Authenticated Cell Cultures [ECACC] no. 84113001), and titrated by both the endpoint dilution method (23) and plaque assay.

Vero cells were cultured in Dulbecco's modified minimal essential medium (DMEM) (Gibco BRL) supplemented with 10% fetal bovine serum (Sigma) and maintained in DMEM without bovine serum.

In vivo and in vitro viral infections of bivalves.

The in vivo effects of OsHV-1 on mussels and oysters were investigated using experimental infections. A total of 60 naive mussels and 60 oysters were inoculated intramuscularly (i.m.) in the posterior adductor muscle with 100 μl of an OsHV-1 suspension (2.7 × 10⁴ copies of viral DNA/μl) at 15°C. Control groups were inoculated with an equivalent volume of filter-sterilized seawater (FSW). All individuals were maintained out of the water for 20 to 30 min before and after the injection. Each treatment group was individually maintained in tanks with aeration. Three experimental challenges were conducted. Cumulative mortalities were monitored for 15 days.

The in vitro effects of OsHV-1 were assayed in mussel and oyster hemocytes extracted from the adductor muscle in mussels and directly from the pericardial cavity in oysters. For each experiment, four pools of hemolymph from five animals (mussels or oysters) were used. The concentration of cells was adjusted to 3 × 10⁶ cells/ml in FSW, and 1 ml of the cell suspension was dispensed in each well of a 24-well plate. The plates were incubated at 15°C for 1 h for settlement and further infected following the procedure previously described by Renault et al. (22). All experiments were performed at 15°C. Cells were sampled at 24 h postinfection (p.i.) to determine the viral titer by quantitative PCR (qPCR). Each experiment was conducted four times.

Detection and quantification of OsHV-1 by qPCR.

OsHV-1 detection and quantification were conducted using standard procedures (24, 25). Briefly, total DNA from the infected hemocytes was isolated using a LEV Blood DNA kit (Promega). Quantitative PCR was performed on an MX3000 thermocycler (Stratagene) with the PCR conditions and primer sequences described in the IFREMER standard operating procedures (25). For quantification, standard curves were obtained using six 10-fold dilutions of a plasmid carrying an OsHV-1 DNA target sequence.

Protein extraction.

Mussel hemolymph was extracted and pooled from 5 mussels and subsequently centrifuged at 3,000 × g for 10 min (4°C) to separate the serum from the hemocytes. The hemocytes were resuspended in 1 ml of homogenization buffer (10 mM HEPES, 250 mM sucrose, 1 mM dithiothreitol [DTT], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF]) supplemented with 1% protease inhibitor cocktail (Sigma; P8340-1 ML) and lysed by passing vigorously through a 25-gauge needle. The resulting lysate was centrifuged at 16,000 × g for 1 h (4°C), and the supernatant was stored at −80°C. The serum was also treated with the protease inhibitor cocktail and stored at −80°C until use.

Protein identification by liquid chromatography-tandem mass spectrometry (LC–MS-MS) analysis.

The samples were methanol-chloroform precipitated, dissolved in 8 M urea-25 mM triethylammonium bicarbonate (TEAB) solution, reduced with 10 mM DTT, and alkylated with iodoacetamide. Samples were digested with trypsin at an enzyme-to-protein ratio of 20:1 (37°C overnight). All the reagents were purchased from Sigma-Aldrich. The proteomic analysis was performed in the proteomics facility of Centro Nacional de Biotecnología.

The peptide samples were analyzed on a nano-liquid chromatography system (Eksigent Technologies; NanoLC-Ultra 1D plus; AB SCIEX, Foster City, CA, USA) coupled to a Triple TOF 5600 mass spectrometer (AB SCIEX, Foster City, CA, USA) with a nano-electrospray ion source.

MS and MS-MS data for each sample were processed using Analyst TF 1.5.1 software (AB SCIEX, Foster City, CA, USA). Raw data were translated into a Mascot generic file (mgf) format and searched against the nrNCBI database with a taxonomic restriction to Metazoa, using an in-house Mascot Server v. 2.4 (Matrix Science, London, United Kingdom). Peptide mass tolerance was set to 50 ppm and 0.2 Da in MS and MS-MS modes, respectively, and 1 missed cleavage was allowed. Typically, accuracy below 10 ppm was found for both MS and MS-MS spectra.

Immunohistochemical analyses.

For in vivo immunohistochemical studies, mussels were injected with 200 μl of a solution containing zymosan (Zym) (1 mg/ml), lipopolysaccharide (LPS) (1 mg/ml), dead Vibrio anguillarum (7.5 × 10⁷ cells/ml), or FSW. Five mussels were used in each treatment, and after 24 h, hemolymph was extracted for further studies, including analyses of myticin expression in the hemocytes (both granulocytes and hyalinocytes).

For in vitro analyses, hemocyte cultures were treated with fluorescent Zym A-TRED particles (Molecular Probes) at a particle-to-cell ratio of 10:1 for 1.5 h at 15°C. Control cells were incubated with FSW. The immunohistochemistry assay of mussel hemocytes was performed using routine procedures: hemocytes were attached to a glass coverslip and incubated overnight (4°C) with anti-myticin C primary antibody (26). Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Life Technologies) was used at a dilution of 1:1,000. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization. The samples were mounted using ProLong antifade reagents for fixed cells (Life Technologies). The images were captured using a TSC SPE confocal microscope (Leica) and processed using LAS-AF (Leica) and ImageJ software.

Flow cytometry analyses.

The distribution of myticin C in different cell populations was assayed in mussel hemocytes by flow cytometry (FACSCalibur; BD). The cell suspensions were adjusted to 3 × 10⁶ cells/ml in FSW, dispensed in 24-well plates (1 ml per well), and incubated for 30 min at 15°C to allow cell settlement. The supernatants were removed, and the cells were washed and fixed with 2% paraformaldehyde (PFA). An anti-myticin C antibody was used to label cells expressing the protein. The density plots were generated using Cell Quest Pro software (BD).

Synthetic myticin C peptides.

A synthetic myticin C mature peptide (Myt-C) (27) and a modified myticin C peptide (Myt-Tat) with a purity of >95% were purchased from New England Peptide (Gardner, MA, USA). The Myt-Tat amino acid sequence contains 40 amino acid residues belonging to the Myt-C peptide plus 13 additional C-terminal amino acid residues corresponding to the cell-penetrating peptide (CPP) of the human immunodeficiency virus type 1 (HIV-1) Tat protein. Myt-C aliquots were diluted in distilled water (dH₂O) supplemented with a protease inhibitor cocktail.

Antiviral activity of myticin peptides against OsHV-1 in oyster hemocytes.

Primary cultures of oyster hemocytes were infected with the OsHV-1 preparation for 1 h at 22°C, as previously described. After infection, the inoculum was removed and the cells were incubated at 15°C with filtered mussel hemolymph, oyster hemolymph, or myticin peptides at a final concentration of 1 μg/ml. At 24 h p.i., samples were taken to evaluate the viral load by qPCR. Two pools of hemolymph from five oysters were used for each assay. The experiment was conducted four times.

Cytotoxicity and antiviral activity of synthetic myticin peptides against human herpesviruses in Vero cells.

First, the cytotoxic effects of Myt-C and Myt-Tat on Vero cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, as previously described (28). Briefly, increasing concentrations of the test samples were added to confluent Vero cells in 96-well plates, with six replicates per concentration. After 72 h of incubation, the MTT solution (0.5 mg/ml) was added, and the plates were incubated for 3 h. The formazan precipitate was dissolved with 2-propanol, and the optical density at 570 nm (OD₅₇₀) was measured using a μQuant spectrophotometer (Biotek Instruments) with a reference wavelength of 620 nm. The resulting cell viabilities after treatment with the test samples were calculated as described by Mosmann (29).

To test the antiviral activity of Myt-C and Myt-Tat against HSV-1 and HSV-2, 96-well plates containing confluent Vero cell monolayers were preincubated (1 h) with 100 μl/well of increasing noncytotoxic concentrations of the peptides (0 to 45 μM), with a replicate number of six wells per test concentration, prior to HSV-1 or HSV-2 infection (10 50% tissue culture infective doses [TCID₅₀]). The plates were incubated at 37°C in a 5% CO₂ humidified atmosphere and observed daily for cytopathic effects (CPEs) using a light microscope. When CPEs were observed in all the virus control wells (approximately 36 h p.i.), the percentage of wells with CPEs was determined for each treatment concentration, as previously described (30). The plates were further incubated for 72 h and then freeze-thawed. The contents of replicated wells treated with the same concentration of peptides, as well as those used as controls, were pooled and titrated by plaque assay, and the viral titers were expressed as PFU per milliliter. Acyclovir (ACV) at concentrations ranging from 0.1 to 10 μg/ml served as a positive control.

The concentration of the sample reducing cell viability by 50% (the mean cytotoxic concentration [CC₅₀]) and that reducing virus-induced CPEs by 50% (mean effective concentration [EC₅₀]) were calculated by regression analysis using the dose-response curves generated from the experimental data. A selectivity index (SI) was calculated for each myticin peptide and virus by dividing the CC₅₀ value by the EC₅₀.

Preparation of nanovesicles carrying the Myt-C peptide and antiviral activity of Myt-C nanosomes.

In order to promote the internalization of Myt-C in the cells, a commercial kit for nanovesicle preparation (Pronanosomes Nio-N Reagent; NanoVex Biotechnologies SL, Spain) was used to encapsulate the synthetic Myt-C peptide into nonionic surfactant nanosomes, following the manufacturer's instructions. Briefly, 250 mg of pronanosomes was mixed by vortexing with 1.5 ml of a 2-mg/ml Myt-C stock solution preheated at 50°C or with an equivalent amount of preheated phosphate-buffered saline (PBS) (negative control). The resulting nanosome solutions were used as stock solutions for preparing the test concentrations of both Myt-C-containing nanosomes (1.01 to 45.0 μM Myt-C plus 0.52 to 16.6 mg/ml nanosomes) and peptide-free nanosomes (0.52 to 16.6 mg/ml of nanosomes) (negative control).

After 1 h preincubation with nanosomes (containing or lacking Myt-C) the cells were infected with 10 TCID₅₀/well of either HSV-1 or HSV-2 and further incubated for 36 h at 37°C in a 5% CO₂ atmosphere. Due to the turbidity of the nanosome preparations, a minor modification of the antiviral assay was used: the supernatants from each treatment concentration were completely collected and pooled for virus titration. The cells were washed gently three times with PBS, left in PBS after the last wash, and examined by microscope for virus-induced CPE. In parallel, the cytotoxic effects of Myt-C-containing and free nanosomes were assessed in Vero cells using the MTT assay in a 96-well plate format, as described previously. After a 72-h incubation, the supernatants were removed and the cells were washed three times with PBS prior to the addition of MTT reagent.

Statistics.

Data from different treatment conditions were compared using unpaired Student t tests or analysis of variance (ANOVA) when possible or the Kruskal-Wallis nonparametric test when necessary, using IBM SPSS-22 Statistics software. The differing means from multiple means comparisons were investigated using the Student-Newman-Keul test and contrasted with Tukey's honestly significant difference (HSD) test or with Dunnett's T3 test.

RESULTS

Mussels are less susceptible to OsHv-1 than oysters.

Differential susceptibility to OsHV-1 infection between mussels and oysters after inoculation was observed. Bivalve mortalities were recorded in the 3 experimental challenges, and differences were also observed. The mortalities among infected oysters started as early as 4 days postinfection and increased until the end of the experiment, reaching high rates (84% ± 7%). In contrast, the mortality of mussels inoculated using the same viral doses and conditions was similar to that of the control animals injected with FSW (Fig. 1A).

FIG 1.

Experimental in vivo and in vitro infections of M. galloprovincialis and C. gigas with OsHV-1. (A) Naive mussels and oysters were injected in the adductor muscle with an OsHV-1 suspension at 15°C. Control groups were inoculated with an equivalent volume of FSW. Cumulative mortality was determined in mussels and oysters infected with OsHv-1 for 15 days. The data are presented as mean values from three independent infections with standard deviations. Infected oysters reached high mortality rates (84% ± 7%), whereas the mortality of infected mussels was similar to that of the control animals. (B) Time course of OsHV-1 replication in primary cultures of mussel and oyster hemocytes. The cells were seeded in a 24-well plate with the addition of 1 ml/well of a 3 × 10⁶-cell/ml hemocyte suspension and incubated at 15°C for 1 h for adhesion. After the incubation, the supernatants were removed and the cells were infected with an OsHv-1 suspension diluted 1:5 in FSW for 1 h at 22°C. After infection, the supernatants were removed and the cells were washed with FSW. Additional hemolymph from oysters and mussels was extracted, centrifuged to eliminate cell components, filtered (0.22 mm), and added to the corresponding wells for incubation at 15°C. The cultures were sampled at 1, 6, and 24 h p.i. to evaluate the viral load by qPCR. The experiment was conducted using duplicated cell cultures. The data are presented as mean values from four independent infections with standard deviations. The viral load in oyster hemocytes was higher than in mussels, and it showed a tendency to increase during the experiment, whereas it did not change significantly in mussel hemocytes.

Next, we determined whether these differences in mortality were related to different abilities of mollusk cells to support OsHV-1 replication. For this purpose, we infected primary cultures of mussel and oyster hemocytes with the same OsHV-1 viral suspension. Significant differences in viral yields were found between oyster and mussel hemocytes (Fig. 1B). The oyster hemocyte cultures had higher viral loads, with values ranging from 2 × 10⁴ to 2 × 10⁵ viral-DNA copies/μl at the end of the experiment. The viral load in the mussel hemocytes was significantly lower (10³ viral-DNA copies/μl) and did not change significantly during the experiment (Fig. 1B).

Mussel hemolymph contains components with antiviral activity.

The differences in mortality and viral replication rates between mussels and oysters suggested that mussel hemolymph could contain compounds conferring resistance to OsHV-1 infection. To evaluate this hypothesis, we determined viral-DNA yields by qPCR in oyster hemocytes incubated with mussel hemolymph and compared these values to the viral-DNA yield of infected oysters incubated with their own hemolymph at 24 h p.i. We observed a significant reduction in the viral-DNA yield of oyster hemocytes treated with mussel hemolymph compared to that of oyster hemocytes incubated with their own hemolymph. There was an approximately 100-fold reduction from 2.9 × 10⁵ DNA copies/μl in mock-treated oyster hemocytes to 1.5 × 10³ DNA copies/μl in oyster hemocytes incubated with mussel hemolymph (Fig. 2).

FIG 2.

Viral loads determined by qPCR in oyster hemocytes infected with OsHV-1 and incubated for 24 h in the presence of oyster (Crassotrea gigas) hemolymph or mussel (Mytilus galloprovincialis) hemolymph. Oyster hemocytes were seeded in a 24-well plate with the addition of 1 ml/well of a 3 × 10⁶-cell/ml hemocyte suspension in FSW and incubated at 15°C for 1 h before infection with OsHV-1. After the incubation, the supernatants were removed and the cells were infected with an OsHv-1 suspension diluted 1:5 in FSW for 1 h at 22°C. After infection, the supernatants were removed and the cells were washed with FSW. New hemolymph from oysters and mussels was extracted, centrifuged to eliminate cell components, and filtered (0.22 mm). Half of the cell cultures were incubated in oyster hemolymph, and the others received mussel hemolymph. The hemocytes were incubated at 15°C and sampled after 24 h to evaluate the viral load by qPCR. Two replicates were used in each of the four independent experiments conducted. The data represent the mean values of a representative experiment with standard deviations. The asterisk indicates statistically significant differences between the treatments (P < 0.05). Oyster hemocytes incubated with mussel hemolymph showed a significant reduction in the viral titer compared with those incubated with their own hemolymph.

To study the composition of mussel hemolymph and to identify the putative compound with antiviral activity, we conducted a proteomic study. Reverse-phase high-performance liquid chromatography (HPLC), followed by high-resolution (triple-time of flight [TOF]) electrospray ionization (ESI)–MS-MS, was carried out. Metabolic and structural proteins, such as actin, were detected in both the serum and hemocytes. Proteins involved in the recognition of foreign particles, such as fibrinogen-related proteins and C1q domain-containing proteins, were also present in these samples. Notably, several peptides derived from myticin, a class of previously reported antimicrobial peptides with known activity against fish viruses, were detected in mussel serum and hemocytes (26) (Table 1).

TABLE 1.

Triple-TOF Mascot search results of M. galloprovincialis hemocytes and serum samples

Sample	Protein or myticina	Species	Accession no.	% coverage	No. of matched peptides/unique sequencesb
Hemocytes	Actin	Diloma radula	AAX51809.1	31	34/5
Hemocytes	Aldehyde DH	Placopecten magellanicus	AAF73122.1	4	7/2
Hemocytes	Aldolase	Anadara sp. strain KJP-2011	ADV41660.1	13	3/1
Hemocytes	α-Enolase	Phyton regius	Q9W7L0.3	13	15/4
Hemocytes	β-Actin	Meretrix meretrix	AEK81538.1	45	74/14
Hemocytes	Calmodulin	Pinctada fucata	AAQ20043.1	22	7/2
Hemocytes	Cytosolic malate DH	M. galloprovincialis	AAZ79367.1	41	37/10
Hemocytes	Elongation factor 1α	M. galloprovincialis	BAD35019.1	27	27/11
Hemocytes	Enolase	Aphanarthrum glabrum	ABB17638.1	16	9/2
Hemocytes	FREP 2/3/6/-	M. galloprovincialis	CBM41041.1	20	10/5
			CBM41042.1	25	14/7
			CBM41045.1	25	7/4
			ADQ55813.1	31	10/6
Hemocytes	Fructose-bisphosphate aldolase	Glottidia sp. strain MR-2009	ACU55147.1	12	6/2
		Haliotis rufescens	AAZ30685.1	11	6/2
Hemocytes	GDP dissociation inhibitor	Geodia cydonium	CAA64439.1	5	4/2
Hemocytes	Glyceraldehyde 3-phosphate DH	Urticina eques	ABM97668.1	11	11/3
Hemocytes	HSP 70	M. galloprovincialis	CAE51348.1	12	14/5
Hemocytes	Phosphoenolpyruvate carboxykinase	M. galloprovincialis	AEP68811.1	23	3/2
Hemocytes	C1q domain-containing protein MgC1q4/5/9/21/24/42/46	M. galloprovincialis	CBX41653.1	25	8/4
			CBX41654.1	41	18/8
			CBX41658.1	26	13/4
			CBX41670.1	31	11/3
			CBX41673.1	20	8/4
			CBX41691.1	34	14/4
			CBX41695.1	31	15/4
Hemocytes	S-Adenosylhomocyshydrolase	Crassostrea ariakensis	ACT35639.1	6	5/3
Hemocytes	Transketolase	Strongylocentrotus purpuratus	NP_001229589.1	4	7/2
Hemocytes	Triosephosphate isomerase	Mytilus edulis	AAT06248.1	36	11/5
		Anadara sp. strain KJP-2011	ADV41659.1	6	3/2
Serum	Actin	Pecten sp.	1101351B	37	42/12
Serum	Calponin-like protein	M. galloprovincialis	BAB60813.1	28	10/8
Serum	Catchin protein	M. galloprovincialis	CAB64664.1	48	96/43
Serum	Elongation factor 1α	M. galloprovincialis	BAD35019.1	13	9/5
Serum	FREP	M. galloprovincialis	CBM41046.1	23	9/6
Serum	Heavy-metal-binding protein HIP	M. edulis	P83425.1	83	587/12
Serum	HSP70	M. galloprovincialis	AAW52766.1	10	7/5
Serum	Myosin heavy chain	M. galloprovincialis	CAB64662.1	38	121/57
Serum	Myosin light chain	Mercenaria mercenaria	1803425A	7	4/1
Serum	Paramyosin	M. galloprovincialis	O96064.1	20	23/15
Serum	C1q domain-containing protein MgC1q4/6/92	M. galloprovincialis	CBX41653.1	6	2/1
			CBX41655.1	65	701/11
			CBX41741.1	18	6/3
Serum	HSP 22	M. galloprovincialis	AEP02967.1	53	20/8
Serum	HSP 24.1	M. galloprovincialis	AEP02968.1	5	2/1
Serum	Superoxide dismutase	M. edulis	CAE46443.1	9	1/1
Serum	Tropomyosin	M. galloprovincialis	P91958.1	64	73/20
Serum	Twitchin	M. galloprovincialis	BAC00784.1	3	11/11
Hemocytes	Myticin A	M. galloprovincialis	P82103.1	13	2/1 MKATILLAVLVAVFVAGTEAHSHACTSYWCGKFCGTASCTHYLCRVLHPGKMCACVHCSRVNNPFRVNQVAKSINDLDYTPIMKSMENLDNGMDML
Serum	Myticin C precursor	M. galloprovincialis	CAM56792.1	22	2/2 MKATILLAVVVVVIVGVQEAQSIPCTSYYCSKFCGSAGCSLYGCYKLHPGKICYCLHCRRAESPLALSGSARNVNEQNKEMVNSPVMNEVENLDQEMNMF
Serum	Myticin A	M. galloprovincialis	P82103.1	13	2/1 MKATILLAVLVAVFVAGTEAHSHACTSYWCGKFCGTASCTHYLCRVLHPGKMCACVHCSRVNNPFRVNQVAKSINDLDYTPIMKSMENLDNGMDML

^aDH, dehydrogenase.

^bMatched peptides are in boldface and underlined in the complete myticin sequences.

Myticin C has antiviral activity against OsHV-1.

To confirm that myticins are constitutively expressed in mussels, we detected these molecules using immunocytochemistry and flow cytometry experiments. We confirmed that myticin C is present in the cytoplasm of normal, unstimulated hemocytes (Fig. 3A). However, not all hemocytes could be labeled using the anti-myticin C primary antibody, suggesting heterogeneity of the cells (Fig. 3A and B). Moreover, the expression of myticin C was not related to external stimuli. After in vitro stimulation of hemocytes with Zym, the number of cells expressing myticin C did not change significantly compared to unstimulated hemocytes (42.4% versus 41.4%) (Fig. 3B). Furthermore, the in vivo treatment of mussels with LPS, Zym, or dead bacteria did not significantly modify the number of granulocytes or hyalinocytes expressing myticin C (Fig. 3C).

FIG 3.

(A) Immunohistochemical localization of myticin C in mussel hemocytes (left). Picture at higher magnification showing the heterogenicity of the mussel hemocytes (right). Scale bar = 0.01 mm. Mussel hemocytes were incubated with an anti-myticin C primary antibody and stained with a fluorophore-conjugated secondary antibody (green fluorescence) and DAPI (blue; cell nuclei). It was observed that myticin C is constitutively expressed in the cytoplasm of mussel hemocytes, although not all hemocytes were positive for myticin C labeling, suggesting heterogeneity of the cells. (B) Distribution of myticin C-positive cells in nonstimulated samples, as determined by flow cytometry using density plots of the fluorescence levels (FL1-H) and the values of complexity registered as side-scattered light (SSC-H). An anti-myticin C antibody and a fluorophore-conjugated secondary antibody were used for labeling cells expressing the protein. The cutoff used in the X parameter FL1-H (log scale) was 37.81. (C) Percentages of Myt-C-positive cells after in vivo stimulation with LPS, Zym, or dead bacteria compared with control individuals (FSW). Hemolymph was extracted after 24 h, and the percentage of granulocytes and hyalinocytes expressing myticin C was determined by flow cytometry using the anti-myticin C antibody and a fluorophore-conjugated secondary antibody. The cutoff used in the X parameter FL1-H (log scale) was 37.81. The number of myticin C-positive cells was not significantly affected by the treatments. Graph represents the mean plus the standard deviation. (D) Confocal image of Myt-C-positive hemocytes engulfing Zym A-TRED particles. The percentages of cells positive and negative for Myt-C expression and/or Zym A-TRED signals are shown in the table. Scale bar = 10 μm.

Another interesting finding during the flow cytometry analysis was the lack of correlation between phagocytosis and myticin C expression: not all the phagocytic cells were labeled with the anti-myticin C antibody. On the other hand, other cells, in addition to phagocytes, expressed myticin C. Therefore, myticin C does not appear to be involved in phagocytosis. Our results also suggest that there is a much more complex functional division in the hemocytes than the classical division into granulocytes and hyalinocytes (Fig. 3D).

Because myticin was constitutively expressed in mussel hemolymph and because we previously reported the antiviral effects of this class of peptides against fish viruses (26), we hypothesized that the naturally occurring resistance of mussels to OsHV-1 infection and the protective effects conferred on oysters by mussel hemolymph against the herpesvirus could be attributed to myticin.

The viral titer in infected oyster hemocytes treated with synthetic myticin decreased by 99.95% at 24 h p.i. This reduction was similar to that recorded for oyster hemocytes incubated with mussel hemolymph (a reduction of 99.39%). However, the combined treatment with myticin peptide plus mussel hemocytes did not synergistically decrease the viral titer. This suggests that the antiviral activity in mussel hemolymph is predominantly due to myticin C, although we cannot rule out the involvement of other molecules in the mollusk antiviral response (Fig. 4).

FIG 4.

Viral load determined by qPCR in oyster hemocytes infected with OsHV-1 and incubated with oyster hemolymph (OH), mussel hemolymph (MH), or the synthetic myticin C peptide diluted in oyster hemolymph (OH + peptide) or mussel hemolymph (MH + peptide) 24 h after infection. Oyster hemocytes were seeded in a 24-well plate with the addition of 1 ml/well of a 3 × 10⁶-cell/ml hemocyte suspension in FSW and incubated at 15°C for 1 h before infection with OsHV-1. After the incubation, the supernatants were removed and the cells were infected with an OsHv-1 suspension diluted 1:5 in FSW for 1 h at 22°C. Then, the supernatants were removed and the cells were washed with FSW. New hemolymph from oysters and mussels was extracted, centrifuged to eliminate cell components, and filtered (0.22 mm). The cell cultures were incubated with OH, MH, OH plus peptide, or MH plus peptide. The hemocytes were incubated at 15°C and sampled after 24 h to evaluate the viral load by qPCR. Two replicates were used in each of the four trials conducted. The data are presented as mean values from four independent experiments with standard deviations. Important reductions in the viral titer were observed both in oyster hemocytes incubated with MH and in those treated with the peptide.

A myticin C-derived peptide inhibits HSV-1 and HSV-2 replication in vitro.

The antiviral effect of myticin C on oyster herpesvirus prompted us to investigate whether this class of molecules has a general antiherpetic activity. For this purpose, we used human HSV-1 and HSV-2 replicating in Vero cells as a surrogate model.

The effects of Myt-C on the viability of Vero cells were evaluated using the MTT colorimetric assay, which is based on the production of formazan by mitochondrial enzymes in viable cells. In the range of concentrations tested, Myt-C was not toxic to the cells because no morphological alterations were detected in the monolayers under a light microscope during the experiment (data not shown). In fact, the highest concentration tested (200 μg/ml, equivalent to 45 μM) resulted in 80% cell viability (Fig. 5A). However, up to this concentration, Myt-C was unable to prevent the appearance of virus-induced CPEs in HSV-infected monolayers (Fig. 5A). The lack of protection against HSV-1 and HSV-2 under these conditions was confirmed by the high viral titers obtained from the cells (on the order of 10⁷ to 10⁸ PFU/ml) (Fig. 5A).

FIG 5.

Cytotoxic effects on Vero cells as determined by the MTT assay (lines) and antiviral effects of myticin peptides against HSV-1 and HSV-2, as determined by the decrease in virus-induced CPE on Vero cells. (A) Cytotoxic and antiviral activities of unmodified Myt-C peptide. (B) Cytotoxic and antiviral activities of Myt-Tat peptide. (C) Cytotoxic and antiviral activities of unmodified Myt-C peptide encapsulated in nanosomes. (D) Cytotoxic and antiviral activities of empty nanosomes. The data are presented as mean values from 3 independent experiments with standard deviations. The percentages of CPE associated with different treatment concentrations within each virus were compared using the Bonferroni post hoc test. The asterisks represent the levels of significance of the differences found for each concentration compared to the untreated (negative) controls. Significant differences are indicated as follows: ***, 0.0001 < P < 0.001; **, 0.001 < P < 0.01; *, 0.01 < P < 0.05. The numbers above the bars represent the virus titers obtained by plaque assay from each pool of replicate treatments within the corresponding concentration (PFU per milliliter).

Next, we used a modified version of the myticin C peptide (referred to as Myt-Tat [see above]) that possesses improved properties of uptake by cells and analyzed its effects against both human herpesviruses. The cytotoxicity (MTT) assay revealed that the modified peptide Myt-Tat was slightly more toxic to Vero cells than Myt-C, which is in agreement with the microscopic observations during the antiviral assay. The main morphological alterations consisted of cell rounding, the appearance of dark nuclear and cytoplasmic inclusion bodies, refringence, and moderate cell detachment (data not shown). These alterations were recorded at the highest concentration tested (32.6 μM, equivalent to 200 μg/ml), which resulted in cell viability of 69.7% using the MTT assay (Fig. 5B).

A significant reduction of virus-induced CPE was observed at relatively low doses of Myt-Tat against both HSV-1 and HSV-2 (Fig. 5B). The EC₅₀s for Myt-Tat were 3.97 ± 0.94 μM (24.35 ± 5.74 μg/ml) against HSV-1 (R² = 0.9871) and 3.09 ± 1.02 μM (18.98 ± 6.27 μg/ml) against HSV-2 (R² = 0.9688). Accordingly, the titers of virus recovered from cells treated with Myt-Tat decreased with increasing concentrations of the peptide, ranging from ∼10⁷ PFU/ml (treatment with 1.01 μM Myt-Tat) to ∼10² to 10³ PFU/ml (treatment with 4.07 μM Myt-Tat). No virus was recovered after treatment with the peptide at a concentration of 8.15 μM or above (Fig. 5B).

One of the best approaches for measuring antiviral activity is the SI, which is calculated as the CC₅₀/EC₅₀ ratio. An SI value greater than 1 indicates specific antiviral activity (31). However, higher SI values are desirable, as they indicate a greater safety margin in antiviral therapy. Because the highest concentration of Myt-Tat used in these assays did not allow calculation of the CC₅₀ value, it can only be suggested that the CC₅₀ would be higher than 32.6 μM. Therefore, the putative SI values for Myt-Tat would be >8.21 against HSV-1 and >10.5 against HSV-2.

The EC₅₀s obtained for ACV, which was used as a positive control in the antiviral activity assay (EC₅₀s, 0.9 μg/ml for HSV-1 and 1.1 μg/ml for HSV-2), are in agreement with previous studies (32, 33) and suggested that both the HSV-1 and HSV-2 isolates used throughout this study are sensitive to acyclovir (EC₅₀ < 2 μg/ml) (34).

The cellular uptake of myticin is required for its anti-HSV effects.

Taking into consideration the reported cell-penetrating activity of the Tat-derived peptide that is C-terminally fused to myticin C in the primary sequence of Myt-Tat and the striking differences between Myt-C and Myt-Tat regarding their anti-HSV activities, we reasoned that the antiviral effects of myticin probably depend on efficient internalization of the peptide, perhaps because the inhibition takes place through the arrest of some intracellular viral replication event. The Myt-C peptide, lacking the Tat-derived cell-penetrating peptide, is not active against HSVs, probably because it cannot be efficiently taken in.

In order to probe this hypothesis, we attempted to potentiate the cell uptake of Myt-C by encapsulating the peptide in commercially available nanovesicles. Under these conditions, the Myt-C peptide recovered its antiviral activity and inhibited both HSV-1 and HSV-2 replication in Vero cells, with EC₅₀s of 5.85 ± 1.24 μM (25.98 ± 5.49 μg/ml) and 5.41 ± 0.95 μM (24.03 ± 4.21 μg/ml), respectively (Fig. 5C). Since the highest concentration of Myt-C conjugated to nanosomes used in the cytotoxicity assays (45 μM Myt-C plus 16.6 mg/ml nanosomes) resulted in cell viability values greater than 50%, it can only be inferred that the CC₅₀ value is higher than 45 μM; thus, the inferred SI values for encapsulated Myt-C were >7.69 and >8.32 for HSV-1 and HSV-2, respectively. The inhibitory effects of Myt-C-containing nanosomes were confirmed by the virus plaque titers obtained from the supernatants of treated cells, which decreased with increasing concentrations of the peptide from ∼10⁷ to ∼10² PFU/ml. No virus was recovered when the infected cells were treated with the Myt-C–nanosome complexes at a concentration of 22.5 μM Myt-C or above, indicating complete suppression of virus replication (Fig. 5C).

The nanosomes themselves did not inhibit virus replication, since 100% virus-induced CPE was observed among HSV-1- and HSV-2-infected cells incubated with concentrations of mock-encapsulated nanovesicles at concentrations equivalent to those used in Myt-C-containing nanosome experiments (0.52 to 16.6 mg/ml of nanosomes) (Fig. 5D). In agreement with this, the viral titers recovered from the supernatants of these cells were similar to those of untreated virus controls (∼10⁷ PFU/ml) (Fig. 5D).

It is worth mentioning that the combination of Myt-C with nanosomes during antiviral evaluation (Fig. 5C) resulted in the most pronounced cytotoxic effect, and the viability of Vero cells treated with the highest concentrations of Myt-C and nanosomes (45 μM and 16.6 mg/ml, respectively) was as low as 62.5%, near the CC₅₀ value. This toxic effect was characterized mainly by a noticeable drop in monolayer confluence from 100% to nearly 70%, although this loss of confluence did not prevent evaluation of the occurrence of virus-induced CPE (data not shown).

DISCUSSION

In vertebrates, the interferon (IFN) response is the major defense mechanism against virus infections. In invertebrates, however, IFN signaling pathways have not been identified, and there is a debate about whether invertebrates possess antiviral immunity similar to the IFN system of vertebrates. Other mechanisms, such as RNA interference or even apoptosis, are considered the main antiviral responses in these animals. However, recent studies have shown that viral infections can trigger inducible and transcriptional responses that have yet to be characterized (35).

In the case of mollusks, knowledge of the antiviral response is even more limited than that of other invertebrates, such as insects. The lack of established mollusk cell lines constitutes a major bottleneck in working with mollusk viruses. Due to their filtering activity, marine bivalves are in contact with a high number of viruses, which commonly reach 10⁷ particles per milliliter in the ocean, including putative pathogens (36). Due to this fact, they should have a constitutive “ready-to-use” repertoire of antimicrobial molecules that can act in dangerous situations. This could be the case with myticins in mussels. Interestingly, M. galloprovincialis, with the exception of some infrequent pathologies, does not appear to be susceptible to diseases (37), in contrast to other bivalves, such as oysters and clams (38–40). In this paper, we have shown that they are resistant to one of the most dangerous bivalve pathogens worldwide, OsHV-1, which does not appear to be a threat for the species.

Although it is possible that the resistance of mussels to OsHV-1 could be due to the lack of specific receptors for the virus, in this study, we confirmed that molecules that are constitutively expressed in naive mussels confer resistance on oysters when their hemocytes are incubated with mussel hemolymph. Although there may be other compounds with potential antiviral activity in mussel serum, in this work, we focused on myticins because they were identified in the proteomic analysis in both serum and hemocytes. Mussel myticins A and B were first described by Mitta et al. (41, 42) as molecules with antibacterial activity against Gram-positive bacteria and with activity against the fungus Fusarium oxysporum and the Gram-negative bacterium Escherichia coli. Myticin C was next identified as an extremely variable antimicrobial peptide with antiviral and antibacterial activities and was also the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples among all invertebrates (26, 27).

It is well known that mussel myticins show high variability at the transcriptional level, but the reasons and the mechanisms underlying this variability are still poorly understood (43–45). The lack of information is even more pronounced at the proteomic level. This study constitutes the first attempt to identify proteins in the hemolymph and immune cells from these animals. The presence of myticin in serum suggests that the molecule is a secreted antiviral compound.

Not all hemocytes express myticins, and the expression of the peptide remained unchanged after immune challenge and was not associated with phagocytosis. It is, therefore, a molecule that is constitutively expressed in mussel cells under normal conditions and is present in hemocytes but also secreted into serum. Until now, nonsecreted compounds with antiviral effects have been identified in mollusk hemolymph in experiments involving hybrid abalone, Haliotis rubra × laevigata, which was infected with abalone herpesvirus 1 (AbHV-1) (46). Prior to this study, the only reported antiviral activity for mollusks was found in fresh filtered Pacific oyster hemolymph, against HSV-1 (47). The EC₅₀ found in that study was 425 μg/ml, which, in light of the current knowledge on antiviral compounds, is too high for the inhibitory activity claimed: EC₅₀s beyond 100 μg/ml are of limited practical value. Thus, in other mollusks, antiviral activity against herpes simplex virus has been observed, but it did not correlate with higher resistance to bivalve herpesviruses (46–48). In this study, for the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks.

The inhibitory activity of myticin against OsHV-1 prompted us to investigate whether it could also inhibit human herpesviruses. The lack of antiviral activity of the unmodified myticin C peptide against human herpesviruses could be related to intrinsic availability properties of the peptide for the cell system employed rather than to specific differences between ostreid and human herpesviruses, for which a common viral ancestor has been suggested (49). An interesting finding of this study was the observation of anti-HSV activity of Myt-C when the cell-penetrating motif derived from the HIV-1 Tat protein was fused to its C terminus. This modification of myticin C appeared to improve its internalization in Vero cells without significantly increasing its cytotoxicity.

In the absence of a Tat-derived cell-penetrating motif, a similar enhancement of anti-HSV activity of Myt-C was recorded when the peptide was encapsulated in nonionic surfactant nanovesicles. The nanovesicles (or nanosomes) are able to fuse with the cell plasma membrane, releasing the peptide into the cytoplasm. Taken together, these results suggest that myticin peptides selectively inhibit an intracellular step of the virus replication cycle instead of directly inactivating free virus particles in the extracellular environment (virucidal activity). The findings presented here highlight a potential use of these antimicrobial peptides from mussels in antiviral therapy, not only for veterinary purposes, but also for human medicine. Further investigation is needed to elucidate the precise mechanisms of antiviral action of myticins.

In conclusion, we still do not know much about the antiviral immune response of invertebrates, and the case of marine bivalves is especially important due to the enormous impact that viral diseases have on shellfish production. Mollusks have the potential to be a source of antimicrobial and antiviral compounds, but most members of the phylum Mollusca have not yet been examined for the presence of these bioactive molecules (36). Myticins are examples of the biotechnological and therapeutic potential of bivalves, which are currently limited to serving as a food supply, and may represent an added value for their culture.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.