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Archives of Microbiology (2018) 200:961–970 https://doi.org/10.1007/s00203-018-1507-1

ORIGINAL PAPER

Polyunsaturated fatty acids from Phyllocaulis boraceiensis mucus block the replication of influenza virus

Ana Rita de Toledo‑Piza1 · Maria Isabel de Oliveira2 · Giuseppina Negri3 · Ronaldo Zucatelli Mendonça1 · Cristina Adelaide Figueiredo2

Received: 11 October 2017 / Revised: 24 March 2018 / Accepted: 27 March 2018 / Published online: 3 April 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

AbstractInfluenza viruses cause worldwide outbreaks and pandemics in humans and animals every year with considerable morbid-ity and mortality. The molecular diversity of secondary metabolites extracted from mollusks is a good alternative for the discovery of novel bioactive compounds with unique structures and diverse biological activities. Phyllocaulis boraceiensis is a hermaphroditic slug that exudes mucus, in which was detected hydroxy polyunsaturated fatty acids that exhibited potent antiviral activity against measles virus. The objective of this study was to evaluate this property against Influenza viruses. Cell viability and toxicity of the mucus were evaluated on Madin–Darby canine kidney (MDCK) cells by MTT assay. Antiviral activity from mucus against influenza viruses was carried out by determination of the virus infection dose and by immuno-fluorescence assays. The crude mucus and its fractions exhibited low cytotoxicity on MDCK cells. A significant inhibition of viral replication, reduced by the order of eight times, was observed in influenza-induced cytopathic effect. In immuno-fluorescence assay was observed a decrease of more than 80% of the viral load on infected MDCK cell treated with mucus and its fractions. The viral glycoproteins hemagglutinin and neuraminidase located on the surface of the virus are crucial for the replications and infectivity of the influenza virus. Some authors demonstrated that lipids, such as, polyunsaturated fatty acids exhibited multiple roles in antiviral innate and adaptive responses, control of inflammation, and in the development of antiviral therapeutics. As corroborated by other studies, hydroxy polyunsaturated fatty acids interfered with the binding of influenza virus on host cell receptor and reduced viral titers. The results obtained indicated that polyunsaturated fatty acids from P. boraceiensis crude mucus and fractions 39 exerted antiviral activity against influenza virus.

Keywords Fatty acids · Antiviral · Influenza · Mollusks

Introduction

Influenza A virus belongs to the family Orthomyxoviridae and cause severe health conditions, often leading to pneumo-nia. The influenza viral genome consists of negative-sense RNA (vRNA) packaged in viral ribonucleoprotein (vRNP) complexes. This enveloped virus, contains eight genomic segments that encode up to 13 proteins, such as, hemagglu-tinin (HA), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), nucleoprotein (NP), non-structural protein 1 (NSP1), non-structural protein 2 (NS2; also known as nuclear export protein, NEP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase basic protein 1-F2 (PB1-F2). The viral envelope formed by a lipid bilayer contains the viral transmembrane proteins: HA, NA, and M2. This virus possess a number of mechanisms that enable it to invade host cells and subvert

Communicated by Erko Stackebrandt.

Cristina Adelaide Figueiredo in memorian.

* Ana Rita de Toledo-Piza artpiza@uol.com.br

1 Laboratory of Parasitology, Butantan Institute, 1500th, Vital Brazil Ave, São Paulo, SP, Brazil

2 Respiratory Infectious Diseases, Adolfo Lutz Institute, 355th, Doutor Arnaldo Ave, São Paulo, SP, Brazil

3 Department of Preventive Medicine, Federal University of São Paulo, 740th, Botucatu St., São Paulo, SP, Brazil

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the host cell machinery in the many steps of their life cycle (Samji 2009).

The World Health Organization (WHO), through the Global Influenza Monitor keeps track of the activity of the disease caused by influenza virus worldwide. Currently, there are three types of influenza viruses, A, B and C, based on the antigenic and genetic differences of the inner proteins and genome structure. Influenza Type A virus is categorized into subtypes based on two surface antigens: hemagglutinin and neuraminidase. The subtypes of influenza virus, type A/H1N1, type A/H3N2 and type B, infect humans, causing massive and rapidly evolving global epidemics. Recently, it was observed that patients infected with the pandemic H1N1 (pdmH1N1) virus developed severe disease and con-sequently died, while those infected with seasonal influenza virus did not develop severe diseases (Zhong et al. 2013; Ding et al. 2017). The severe “Spanish flu” in 1918, caused by the H1N1 strain, was the worst pandemic in recorded his-tory and resulted in approximately 50 million deaths world-wide (Taubenberger et al. 2001). During the year 2009, 214 countries reported cases of H1N1 pandemic influenza with more than 18,000 deaths (Jin and Mossad 2012).

Influenza A (H1N1) virus remained a great health issue, and there are few treatment options. Vaccination remains the primary method for prevention of influenza. However, vaccines only protect against antigenically related strains and exhibited limited efficacy. Antigenic drift, or mutations mainly of the segments encoding the surface proteins and promote evasion of the host immune system. The high diver-sity of potential emerging zoonotic and pandemic viruses become difficult to select the right strains for vaccine pro-duction, and consequently allows a slightly varied virus to re-infect the population (Nachbagauer and Krammer 2017). The antiviral drugs approved for the influenza virus pre-vention and therapy are the viral M2 protein, Amantadine® (Symmetrel) and Rimantadine® (Flumadine), or the neu-raminidase inhibitors, Zanamivir® (Relenza) and Oseltami-vir Carboxylate® (Tamiflu). M2 is tetrameric proton chan-nel activated by acidic pH, which is important for genome unpacking during virus entry (Tam et al. 2013). The M2 channel transports protons from the vacuolar space into the interior of the virion, while Neuraminidase inhibitors act as a competitive inhibitor of the activity of the viral neurami-nidase enzyme. The M2 ion-channel inhibitors is effective only against type A viruses (Pielak and Chou 2010), while the neuraminidase inhibitors, is effective against both type A and B viruses.

Depending on the way, as was detected, the levels of anti-viral resistance are classified as genotypic, phenotypic and clinical resistance (Nitsch-Osuch and Brydak 2014). These antiviral drugs facing drug resistance in new strains (Dolin 2011; Longtin et al. 2011; Hurt et al. 2012). Oseltamivir and Zanamivir are the two main neuraminidase inhibitors

used for the treatment of Influenza. The clinical use of neu-raminidase inhibitor oseltamivir increased substantially during the recent H1N1 pandemic. Oseltamivir resistance was identified in non-pandemic influenza viruses, as well as H1N1 pandemic influenza A viruses (Shankaran and Bear-man 2012). The most common mutation associated with oseltamivir resistance is the amino acid change H275Y in the neuraminidase (NA) protein (Sheu et al. 2008; Renaud et al. 2011). Among 2007–2008, the oseltamivir-resistant strain of the seasonal H1N1 virus was efficiently transmitted, causing a widespread epidemic of drug-resistant influenza (Ruangrung et al. 2016). In 2011, were reported 570 cases of oseltamivir-resistant influenza A (H1N1) around the world (Gioula et al. 2011; Storms et al. 2012).

Molluscan species biosynthesized secondary metabolites that are crucial constituents for its defenses against viruses. The secondary metabolites are extracted from the innate immune system, which is provided by physical barriers, such as, shell, skin, mucus, and epithelium. These exudates exhibited a variety of immune mechanisms that include antiviral compounds that exhibit diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens (Dang et al. 2015). Several bio-active molecules extracted from mollusks exhibited poten-tial pharmaceutical or industrial applications. Besides, this can be used as a source of antibacterial and antiviral drugs. However, most members of the phylum Mollusca have not studied for the search of bioactive compounds (Novoa et al. 2016). The exudate extracted of marine mollusks from the family Muricidae are used in traditional medicines for thou-sands of years. The brominated indoles extracted exhibited anti-inflammatory, anti-cancer and steroidogenic activity, while the choline esters showed muscle-relaxing- and pain-relieving properties (Benkendorff et al. 2015). Fatty acids were reported in mollusc species (Zhukova 2014). In gen-eral, mollusks are a source of lipid bioactive compounds, which produce pharmaceutical applications (Benkendorff 2010). The omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid, 20:5n-3, and docosahexaenoic acid, 22:6n-3, are used in the treatment of cardiovascular disease and other chronic diseases, because reduce triacylglycerol and cholesterol levels and exhibited anti-inflammatory and anti-cancer activity (Simopoulos 2008).

The terrestrial slug Phyllocaulis boraceiensis (Thomé, 1972) secrete visco-elastic mucus. This exudate acts as an adhesive and lubricant, and enables the creatures to adhere to, and glide over, all types of surfaces including rough or potentially hostile terrain. Besides this, mucus prevents infection and facilitate healing (Zhukova 2014). The potent antiviral activity of P. boraceiensis crude mucus and its frac-tions on Vero cells infected with measles virus were attrib-uted to polyunsaturated fatty acids (Toledo-Piza et al. 2016). Some studies demonstrated the antiviral action of this sort

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of molecule on enveloped viruses. The aim of this study was to evaluate the antiviral action of crude mucus from P. boraceiensis and its fractions, which contain polyunsatu-rated fatty acids against the enveloped influenza viruses.

Materials and methods

Collection and solubilization of Phyllocaulis boraceiensis mucus

In this study, 20 specimens of the terrestrial slug Phyl-locaulis boraceiensis were farmed in the vivarium of the Malacology Laboratory at the Butantan Institute, São Paulo. The Ethics Committee on Animal Use at the Butantan Insti-tute (CEUAIB) declared that this study does not involve the creation and/or use of animals in the phylum Chordata (I-1133/13).

The animals were kept in plastic boxes containing soil “in natura” and a screened cover, in a pollution-free laboratory environment with the temperature controlled at 24 °C with 85% relative humidity. All specimens were fed every 2 days with small amounts of lettuce and the boxes were cleaned every 2 days.

Before the collection of mucus, the animals were trans-ferred to a large Petri dish and nebulized with distilled water (Li and Graham 2007). The removal of the mucus was stimulated by mechanical processes that did not cause death or damage to the specimens (Pakarinen 1994). The animals were cleaned for 5 min on the smooth surface of the Petri dish containing a thin layer of saline solution (0.06% NaCl). The addition of this solution to the collection plates facilitated the release and removal of mucus, which was removed with the aid of a spatula and transferred to a storage container. The solubilization/dissolution process required organic solvents, such as ethanol and methanol (Toledo-Piza et al. 2013).

An amount of 20 mL of crude mucus were collected. After solubilization process and liophilization, an amount of 500 µg of crude mucus in pounder were tested. The P. bora-ceiensis mucus samples were concentrated using a mem-brane of regenerated cellulose (Ultracel®-3H-AmiconUltra, Millipore). The supernatant, obtained after filtration was cleaned using a 2D Clean Up Kit (GE Healthcare). This solution was used in all experiments.

Fractionation of Phyllocaulis boraceiensis mucus

Stored samples of P. boraceiensis mucus were centrifuged at 1000g for 10 min and filtered through a sterilizing 0.22 µm membrane. For the fractionation of the mucus, a gel filtration column was used (Superdex™75, GE Healthcare) coupled

to a high pressure chromatograph (Akta Purifier, GE Health care).

Analysis of fractions 39 and 50 of Phyllocaulis boraceiensis mucus using fourier transform infrared spectrometry (FT‑IR)

FT-IR spectra were recorded at room temperature (ca. 25 °C) using a Bomem spectrometer by scanning over the frequency range of 4000–400 cm− 1 at a resolution of 5 cm− 1. Fractions 39 and 50 were dissolved in methanol and analyzed using potassium bromide pellets.

Analysis of fractions 39 and 50 of Phyllocaulis boraceiensis mucus using reversed phase high‑performance liquid chromatography‑diode array‑electrospray ionization mass spectrometry/mass spectra (HPLC‑DAD‑ESI‑MS/MS)

HPLC-DAD-ESI-MS/MS analysis was conducted on a DAD-SPD-M10AVP Shimadzu system equipped with a photodiode array detector coupled to an Esquire 3000 Plus system (Bruker Daltonics), which consisted of two LC-20AD pumps, an SPD-20A diode array detector, a CTO-20A column oven and an SIL 20AC auto injector (Shimadzu Corporation Kyoto, Japan). The mass detector was a quadrupole ion trap equipped with an atmospheric pressure ionization source through an elec-trospray ionization interface, which was operated in full scan MS/MS mode. All the operations, acquisition and data analysis were controlled by CBM-20A software. The mobile phase was filtered through a 0.45 mm Millipore filter and degassed prior to use. Fractions 39 and 50 were dissolved in methanol:water (80:20 v/v) and filtered through a 0.45 µm PTFE filter, prior to injecting 50 µL into the HPLC system. The peaks were also monitored by diode array detection at a wavelength of 270 nm. The mobile phase consisted of two solvents: eluent A (0.1% aq. formic acid in Milli-Q water) and eluent B (methanol 0.1% aq. formic acid). The constituents were separated using a reverse phase, Phenomenex Gemini C-18 (250 × 4.6 mm, 5 μm) col-umn connected to a guard column. The elution started with 10% B in A; 5 min − 20% B in A;15 min − 30% B in A; 20 min − 40% B in A; 25 min − 50% B in A; 30 min − 60% B in A, 35 min − 70% B in A; 40 min − 80% B in A; 50 min − 100% B in A and finally back to the initial conditions (10% B in A) to re-equilibrate the column prior to another run. The flow rate was kept constant at 1.0 mL min− 1 and the temperature of the column was maintained at 40 °C. The ionization con-ditions were adjusted as follows: electrospray ionization was performed using an ion source voltage of 38 V and a capillary offset voltage of 4500 V. The full scan mass acquisition was performed using electrospray ionization in positive ion mode by scanning from m/z 100–1200 Da. Helium was used as the collision gas and nitrogen as the nebulizing gas. Nebulization

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was aided with a coaxial nitrogen sheath gas provided at a pressure of 27 psi. Desolvation was enhanced using a counter current nitrogen flow set at a flux of 7.0 l/min and a capillary temperature of 320 °C. The data-dependent MS/MS events were assessed based on the most intense ions detected in full-scan MS. The maximum accumulation time of the ion trap and the MS numbers to obtain the MS average spectra were set at 30 and 3 MS, respectively. All peaks were defined through the interpretation of ESI-MS and ESI-MS/MS spectra and by comparison with the literature data (Murphy 2014).

The farm of Phyllocaulis boraceiensis, collection, solubi-lization, fractionation and analysis of its mucus using infra-red and HPLC-DAD-ESI-MS/MS was described previously (Toledo-Piza et al. 2016) and reproduced here.

MTT assay

MDCK (Madin–Darby canine kidney ATCC CCL 34) cells were grown in Leibovitz’s L-15 growth medium supple-mented with 10% inactive fetal bovine serum (FBS) and 20 mM of l-glutamine (Invitrogen, EUA). Detailed proce-dure was reported by Toledo-Piza et al. (2016).

Determination of the virus infectious dose

MDCK cells (2.0 × 104 cells/mL) were cultivated in 96-well plates in Leibovitz’s L-15 growth medium supplemented with 10% FBS and incubated at 37 °C. Influenza A strain were quantified by medium tissue—culture infections with multiplicity of infection (MOI-1) on cell cultures. Detailed procedure was reported by Toledo-Piza et al. (2016).

Determination of antiviral activity against infected cell cultures using fluorescent antibodies

MDCK cells were incubated with 60, 120 and 180 ng/mL of P. boraceiensis mucus and fractions 39, 40 and 49 dur-ing 1 h at 37 °C followed by washing with L-15 medium and incubation with influenza virus (MOI-1). The cells were incubated with 10 µg/mL of mouse monoclonal antibodies influenza A virus (Millipore, EUA) in 50 µL of phosphate-buffered saline (PBS) + bovine serum albumin (BSA) 1% solution at 4 °C overnight. The antiviral activity was meas-ured on infected cells, in which the production of influenza A virus fluorescent antigens was blocked. Detailed proce-dure was reported by Toledo-Piza et al. (2016).

Results

Biological experiments were carried out using fractions 39 and 50, which were analyzed using Fourier trans-form infrared spectrometry (FT-IR) and reversed phase

high-performance liquid chromatography-diode array-electrospray ionization mass spectrometry/mass spectra (HPLC-DAD-ESI-MS/MS).

The Fourier transform infrared spectrum of fraction 39 showed absorption bands at 3368, 3205, 3010, 2955, 2917, 2849, 1629, 1517, 1463,1402, 1378, 1296, 1059 and 1038 cm− 1. The large and strong band at 3368–3205 cm− 1 indicated the presence of OH and COOH groups; the bands at 2599, 2917 and 2849 cm− 1 (C–H, asymmetric and sym-metric stretching vibration, respectively) indicated the presence of long carbon chains. The ratio of the peak area CH=CH (3010–2950 cm− 1) versus CH2 (2955–2917 cm− 1) denoted the unsaturation index of fatty acids (Wu and He 2014). A strong peak at 3010 cm− 1 is originated from C–H stretching modes. The band at 1629 cm− 1 was attributed to C=O stretching vibrations conjugated with double bonds (C=C) adjacent to a carbonyl group. The bands at 1517 and 1463 cm− 1 were attributed to stretching of double bonds and CH2 bending, respectively; the strong bands at 1059 and 1038 cm− 1 were attributed to stretching vibrations of C–O and C–H bending. The C–O stretching vibrations in alco-hols produce a strong band in the 1260–1000 cm− 1 region of the spectrum (Kiefer et al. 2010; Shapaval et al. 2014). For fraction 50, the main absorption band was detected at 1731 cm− 1, attributed to stretching of C=O groups of ketones or esters (Shapaval et al. 2014; Wu and He 2014). Therefore, the main difference among the IR spectra of fractions 39 and 50 was the presence of band at 1731 cm− 1 assigned to the C=O stretch, the other bands exhibited minor intensity.

The technique of electrospray ionization (ESI) is used for analyses of polyunsaturated fatty acids (Murphy 2014). In reversed phase liquid chromatography, the elution order of fatty acid derivatives is mainly affected by the number of carbon atoms and the number of unsaturated bonds in the fatty acid chain (Hsu and Turk 2008; Bollinger et al. 2013). Many different types of hydroxy fatty acids are formed as intermediates of fatty acid biosynthesis and metabolism. Table 1, published previously (Toledo-Piza et al. 2016), summarizes the following information on the peaks observed during RPHPLC-ESI-MS/MS analyses: retention times (Rt), MS spectral data for sodiated and protonated molecules and proposed structures for polyunsaturated fatty acids 1–5. The sodium adduct ions, [M + H + Na]+ possess 23 Da above the proposed quasi-molecular ion in first-order mass spectra obtained with ESI in positive ion mode (Trufelli et al. 2011; Xie et al. 2012; Wilson et al. 2015).

In fraction 39 and 50, compounds 1–3 represent a homol-ogous series of compounds, in which the molecular mass (MM) differed by 28 Da. Compound 5 found only in frac-tion 50 possess 112 Da (8 CH2) more than compound 3. The fragmentation pattern of compounds 1–3 is very similar and the product ions obtained following collisional activation

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of sodiated adduct (M + H + Na)+ produce abundant frag-ments at m/z 270 (NaC15O3H20)+, attributed to the product ion (NaC15O3H20)+ corresponding to the loss of 225 Da and at m/z 298, which possess 28 Da more than fragment ion at m/z 270. This fragmentation pattern indicated that com-pounds 1–5 are polyunsaturated fatty acids (Murphy 2014).

The ESI-MS spectrum of compound 1, exhibited sodiated molecule at m/z 495 (Table 1), indicating MM corresponding to 472 g/mol, and was assigned as hydroxy-hentriacontap-entaenoic acid. Fatty acids possess many different complex species and the determination of the position of the double bond is not possible using only mass spectral fragmentations data (Trufelli et al. 2011; Thomas et al. 2014). Generally, for polyunsaturated fatty acid derivatives, all double bonds have the cis (Z) configuration. The ESI-MS spectrum of compound 2 (Table 1) exhibited a sodiated molecule at m/z 523, corresponding to MM of 500 g/mol. Compound 2 was assigned as hydroxy-tritriacontapentaenoic acid. For com-pound 3 (Table 1), the ESI-MS spectrum showed a sodiated molecule at m/z 551, corresponding to an MM of 528 g/mol, and was assigned as hydroxy-pentatriacontapentaenoic acid. For compound 4, the ESI-MS spectrum exhibited a sodiated molecule at m/z 413, which correspond to MM of 390 g/mol. After MS/MS experiments, the precursor ion at m/z 413 produced abundant fragment ion at m/z 301, attributed to the loss of 112 Da (8 CH2). Compound 4 was assigned as hydroxy-pentacosatetraenoic acid.

Compounds 2 and 3 were the main constituents present in fraction 39, while compounds 1 and 4 were found at low contents. In fraction 50, compounds 1–4 were found in low contents. Compound 5 is the main constituent pre-sent in fraction 50, which probably is responsible for the strong absorption band at 1731 cm− 1 in the infrared spec-trum, attributed to (C=O) stretching. Compound 5 exhibited sodiated molecule at m/z 685 and protonated molecule at

m/z 663, corresponding to MM of 662 g/mol. The MS/MS spectrum of the precursor ion at m/z 663 showed product ions at m/z 607 (80), m/z 551 (100) and m/z 495 (70); in which each of these fragment ions correspond to the loss of 56 Da (4 CH2). Compound 5 was tentatively assigned as oxo-pentatetracontaheptaenoic acid. The proposed structure for compounds 1–5 need to be confirmed through NMR 1H and 13C studies.

The crude sample of mucus and its fractions (39, 40 and 49) were tested in MDCK cells infected with influenza viruses. The results obtained by MTT colorimetric method showed that P. boraceiensis mucus was not toxic to MDCK cells. The lethal concentration (IC50%) was 41.0, 73.2 and 92.6 µL, when cells were treated with crude mucus, fraction 39 and fraction 49, respectively. MDCK cells were infected with different dilutions of influenza virus H1N1. Replication of viruses were observed by morphologic changes, known as cytopathic effects (CPE), which can be observed easily in unfixed, unstained cells often clarified by electron micros-copy. The CPE generated by infection on MDCK cells was observed daily. The virus infectious dose was determined by the highest dilution of virus capable of inducing CPE on MDCK cells infected and treated with mucus. The antivi-ral effect of polyunsaturated fatty acid on virus growth was determined by the difference among the CPE observed on infected MDCK cells versus infected MDCK cells treated with crude mucus and its fractions (39, 40 and 49). As can be seen Fig. 1, the crude mucus and the fraction 39 inhibited 8 times the viral growth of H1N1 virus, when compared to uninfected negative control.

Studies of viral replication using confocal microscopy and monoclonal antibody influenza A virus indicated that viruses produce double-stranded RNA, that is essential for the induction of the host immune response (Son et al. 2015). The activity of potential antiviral compounds can

Table 1 RPHPLC-DAD-ESI-MS/MS analyses of mucus fraction 39 and 50: retention times (Rt), MS data for protonated molecules and proposed structures—Published previously (Toledo-Piza et al. 2016)

Retention time MS data protonated molecules Proposed structures

1 48.7 [M + Na]+-495MM—472—C31O3H52MS/MS—270 (100), 298 (20), 242 (20)

Hydroxy-hentriacontapentaenoic acid

2 49.4 [M + Na]+523MM—500—C33O3H56MS/MS—270 (100), 298 (70)

Hydroxy-tritriacontapentaenoic acid

3 50.0 [M + Na]+ 551MM—528—C35O3H60MS/MS—298 (100)

Hydroxy-pentatriacontapentaenoic acid

4 53.3 [M + Na]+ 413MM—390—C25O3H42MS/MS—301 (100)

Hydroxy-pentacosatetraenoic acid

5 53.6 [M + Na]+ 685[M + H]+ 663MM—662—C45O3H74MS/MS—551 (100), 607 (80), 495 (70)

Oxo-pentatetracontaheptenoic acid

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be measured by their ability to inhibit a stage or enzy-matic activity in viral replication cycle. Immunofluo-rescence assay is recommended for rapid visualization of respiratory viruses, including influenza virus, using specific monoclonal antibody. The procedure involved the treatment of infected cells on ice with the non-ionic detergent Triton X-100. Membrane parts enriched in cholesterol and sphingolipids did not solubilize by this procedure because are detergent-resistant membrane (Veit et al. 2013). Infection process, virus entry and viral replication determined by immunofluorescence assay rely on the specificity of fluorescent-labeled antibodies against their corresponding antigens within a cell. The antiviral activity was determinate by the proportion of infected cells, in which the production of influenza A virus fluorescent antigens was blocked. In Fig. 2a can

be seen the uninfected negative control. Figure 2b shows MDCK cells infected with influenza virus H1N1 and influenza A-labeled anti-antibody (green). Uninfected cells are marked in red with Evans blue. Figure 2c shows infected MDCK cells treated with crude mucus of P. boraceiensis, in which can be observed marked decrease in the number of fluorescent cells. Figure 2d–f exhibit the results of infected MDCK cells treated with semi-purified mucus fractions 39, 40 and 49. In the immuno-fluorescence assay was observed a decrease of more than 80% of the viral load on the infected MDCK cell cultures treated with mucus. Thus, the antiviral effect was signifi-cant on infected MDCK cells treated with crude mucus and fraction 39. The smaller number of cells observed on the infected and untreated MDCK cell cultures was attributed to the time of collection, because most of the

Fig. 1 MDCK cells were pretreated with 2% of P. bora-ceiensis mucus and the three fractions obtained by chroma-tography in a chromatograph “AKTA Purifier”. The plate was examined every day by observ-ing the appearance of cytopathic effect. After 72 h, the culture was stained with crystal violet (0.2%). Positive control virus (CV); crude mucus; fraction 39; fraction 40; fraction 49. The results are representative of three experiments

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cell carpet was destroyed by cytopathic effect. On the other hand, the maintenance of the cell carpet for longer time on infected MDCK cells treated with the fractions 39 and 49 of mucus, indicated that polyunsaturated fatty acids controlled, at least partially, the infection caused by influenza virus.

Discussion

Influenza A infection begins with virus entry and trans-portation within the nucleus cells. The influenza virus life cycle are divided into the following steps: entry into

Fig. 2 Fluorescence in MDCK cells infected with influenza virus (H1N1) in inverted microscope, Influenza-labeled anti-antibody (green) and Evans blue (red). MDCK cells not infected control (a); MDCK cells infected with 1 moi of H1N1 (b); infected and treated cells with crude mucus of P. boraceiensis (c); infected cells treated with fraction 39 (d); infected cells treated with frac-tion 40 (e) and infected cells treated with fraction 49 (f). The slides were analyzed with laser confocal microscope LSM 510 META (×200 magnification)

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the host cell; entry of viral ribonucleoproteins (vRNPs) within the nucleus; transcription and replication of the viral genome; export of the vRNPs from the nucleus; and assembly and budding at the host cell plasma membrane (Samji 2009). mRNA expression and virus titers rapidly increased after infection (Teissier et al. 2011). In response to excessive viral load, host cells produce cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-α). The activation of antiviral interferons (IFNs) induced innate immune response to viral infections. Accumulation of IL-6 and TNF-α is responsible for the pathogenesis and severity of influenza virus infection, which can cause severe secondary pneumonia in the lung (Shi et al. 2017).

Virus entry depends on viral surface proteins and host cell receptors. Most cellular receptors are surface proteins that developed many functions. The viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) located on its membrane are crucial for the replications and infectivity of the influenza virus (Fiers et al. 2004). Viral interaction with cellular membrane compartments during their replication, induces cytoplasmic membrane structures, in which genome replication and assembly occurs (Zhong et al. 2013; Shi et al. 2017). There are multiple innate immune mechanisms evolved to modify the lipid composition of cellular and viral membranes to inhibit virion fusion of enveloped virus. HA protein that recognizes glycoprotein on the host cell membrane, exert a fundamental role for cell entry of virus particles. Virus need to cross membranes for cell entry and exit, which in enveloped virus occurs by membrane fusion. HA protein catalyses the receptor binding and membrane fusion (Veit et al. 2013), playing an important role in the pathogenicity of influenza virus (Zhong et al. 2013). Viral replication occurs exclusively inside the respective host cell. Therefore, the inhibiting the adsorption of HA proteins to host cells is an effective way to prevent influenza A infection (Zhong et al. 2013).

Lipids, among them, polyunsaturated fatty acids exhib-ited multiple roles in antiviral innate and adaptive response control of inflammation, and potentially in the development of antiviral drugs (Schoggins and Randall 2013). Polyun-saturated fatty acids (PUFA) are essential for life because they are important determinants of physical and chemical properties of membranes (Hulbert et al. 2014), and can exert a potential antiviral effect (Shi et al. 2017). Mollusks contain a great variety of PUFA (Zhukova 2014). Very long-chain fatty acids known as demospongic acids were detected in Nudibranch mollusks, which are not protected by a shell (Zhukova 2014). The terrestrial snails, Helix pomatia and Helix nemoralis, contain a high content of polyunsaturated fatty acids with more than 20 carbon atoms (Miletic et al. 1991). The lipid composition of snails and slugs, belong-ing to the phylum Mollusca, exhibited the polyunsaturated fatty acids, linoleic (18:2n-6), arachidonic acids (20:4n-6),

linolenic (18:3n-3), eicosapentaenoic acids (20:5n-3) and docosapentaenoic acid (22:5n-6) (Zhu et al. 1994). The lipid composition of the far-east bivalve mollusk Spisula sachalinensis exhibited the presence of 46 fatty acids of various structures with different localizations of double bonds, including 12 saturated, 15 unsaturated, and 19 poly-unsaturated ones (Tabakaeva and Tabakaev 2017). Fraction 39 isolated from mucus of Phyllocaulis boraceiensis, con-tain polyunsaturated fatty acids (Toledo-Piza et al. 2016). Their identification was also based on a liquid chromatog-raphy–mass spectrometry (LC-MS/MS) analysis developed by (Arita 2012).

The influenza virus infections can be inhibited during their entry at the step of fusion with cellular membranes. Alterations of membrane lipid composition can block viral release and entry (Lorizate and Kräusslich 2011). In envel-oped virus infection, the major lipid-dependent interaction is the fusion of their lipid bilayer envelope with a cellular mem-brane to release the viral genome into the cytoplasm (Schog-gins and Randall 2013). Fatty acids act as fusion inhibitors of membranes. Arbidol (ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate) demonstrated antiviral activity against a number of enveloped and non-enveloped viruses (Boriskin et al. 2008), acting on different aspects of the fusion process. Arbidol, approved for influ-enza A and B virus treatment in Russia and China, modu-late fusion protein interaction with phospholipids to prevent protein conformational changes required for fusion (Teissier et al. 2011).

As observed in the previous reported study (Toledo-Piza et al. 2016), fraction 39, containing the hydroxy-tritriaco-ntapentaenoic acid and hydroxy-pentatriacontapentaenoic acid, exhibited the best antiviral activity. A similar com-pound, 10S,17S-dihydroxydocosahexaenoic acid markedly attenuated influenza virus replication, interfering with bind-ing of receptor host nuclear export factors to influenza virus RNAs, that prevented viral RNA export from the nucleus to the cytoplasm. This compound acts as a potent inhibitor of virus replication, reducing viral titers (García-Sastre 2013; Morita et al. 2013). 15-Hydroxyeicosapentaenoic acid and 15-hydroxyeicosatrienoic acid exhibited anti-inflammatory properties and antimicrobial potency in treatments for Gram-positive bacterial infections (Desbois and Lawlor 2013). According to Tam et al. (2013) the bioactive lipid profiles can be used as useful biomarkers to indicate the immuno-logical status of an active infection and the host response to influenza. 9-Hydroxyoctadecadienoic acid exhibited a proin-flammatory activity, while 13-hydroxyoctadecadienoic acid acted as anti-inflammatory. Virus entry and viral replication are the key steps for influenza virus infection. The experi-mental assays indicated hydroxy-tritriacontapentaenoic acid and hydroxy-pentatriacontapentaenoic acid inhibited the

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viral absorption on MDCK cells, suppressed viral replication and protected MDCK cells from viral damage. Thus, these polyunsaturated fatty acids showed activity against influ-enza virus, probably, acting as inhibitor of virus entry and viral replication, which was observed through the reduction cytopathic effects and viral titers. These observations was corroborated by other authors that demonstrated the antiviral activity of polyunsaturated fatty acids against influenza virus (García-Sastre 2013; Morita et al. 2013; Tam et al. 2013).

The study of bioactive molecules capable of acting as antiviral agents is very important, due to high diversity of potential emerging zoonotic and pandemic influenza viruses. Recent studies demonstrated the importance of the search for new antiviral compounds extracted from mollusks. For P. boraceiensis mucus, there are few studies about chemical composition and antiviral activity. The antiviral effect was attributed to polyunsaturated fatty acids. As was corrobo-rated by other author, hydroxy polyunsaturated acids can block the binding of virus on-host cell receptor and pen-etration within cell, preventing the viral replication. Thus, hydroxy polyunsaturated fatty acids from mucus could act as potent antiviral agents in the control of epidemics and pandemics caused by influenza virus.

Funding This study was funded by São Paulo Research Foundation (FAPESP—2012/22906-9 and 2012/22555-1).

Compliance with ethical standards

Conflict of interest The authors declare that there are no conflicts of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

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