In vitro evaluation of antiviral and virucidal activity of a high molecular weight hyaluronic acid

Claudio Cermelli 2 Alessandro Cuoghi 2 Monica Scuri 1 Clotilde Bettua 2 Rachele G Neglia 2 Andrea Ardizzoni 2 Elisabetta Blasi 1 Tommaso Iannitti 0 Beniamino Palmieri 3 0 Department of Biological and Biomedical Sciences, Glasgow Caledonian University , Glasgow , UK 1 Department of Laboratories, Pathological Anatomy and Legal Medicine, University of Modena and Reggio Emilia , Italy 2 Department of Public Health Sciences, University of Modena and Reggio Emilia , Modena , Italy 3 Department of General Surgery and Surgical Specialties, University of Modena and Reggio Emilia , Modena , Italy Background: hyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour serum and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the in vitro antiviral activity of a high molecular weight HA. Methods: the MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes Simplex Virus-1, Mumps Virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays. Results: the most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4 mg/ml, and a reduction of 3.5Log and 2Log, at 2 mg/ml and 1 mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1 mg/ml and 1Log at 4 mg/ml and 2 mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4 mg/ml. Herpes Simplex Virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle. Conclusions: the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis. - Introduction Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan which consists of alternately repeating D-glucuronic acid and N-acetylglucosamine units. A huge variety of HAs, with different molecular weights, has been described, probably retaining distinct physicochemical and biological properties. HA is naturally present throughout all mammalian systems, especially synovial fluid, vitreous humour serum and many connective tissues [1]. Moreover, HA is found intercellularly in connective tissues, such as skin, combined with proteins and chondroitin sulphate, where it fulfills important functions involved in tissue structure maintenance, moisture and lubrication [2]. Initially introduced in clinical practice as wound healing promoter, HA is currently used in many medical and cosmetic fields. Some examples of HA applications include eye drops for kerato-conjunctivitis, intra-articular injections for osteoarthritic joint pain, irrigations for bladder and vaginal chronic inflammatory disorders, tracheobronchial aerosolization for asthma, oral solutions for mouth care or for oesophageal-reflux and gastritis. Besides, HA is commonly used for cosmetic interventions, as a filler to counteract ageing and facial lipoatrophy, especially in HIV patients [3]. There is evidence showing the ability of HA to interfere with viral replication in vitro. In particular, the replication of Herpes Simplex Virus type 2 [4], Respiratory Syncytial virus [5] and retroviruses [6] is inhibited by HA, while the Adenovirus (ADV) one results enhanced [7]. Such limited and apparently controversial data demand further investigations in order to better understand the HA biological properties. In this study we investigated the in vitro effects of a high molecular weight HA against a wide group of viruses covering a large spectrum of structural features and replication strategies: ADV-5, Coxsackievirus B5 (COXB5), Herpes Simplex Virus type 1 (HSV-1), Human Herpesvirus-6 (HHV-6), Influenza Virus A/ H1N1, Mumps Virus (MV), Porcine Parvovirus (PPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). We observed an antiviral activity against COXB5, HSV-1, MV, PPV and Influenza Virus encouraging the use of such compound as a medical tool in specific clinical circumstances. Materials and methods Hyaluronic Acid A high molecular HA (1.800 KD) in powder (IBSA, Istituto Biochimico SA, Lugano, CH) was used. It was dissolved in Minimum Essential Medium (EMEM) at 8 mg/ml solution and sterilized by filtration through 0.45 m filters. Cells and Viruses The following cell lines were used to cultivate the different viruses: two monkey kidney lines, VERO cells for ADV-5, COXB5, HSV-1, and MV and MARC145 cells for PRRSV; the human T-leukaemia lymphoblast line JJHAN for HHV-6; the canine kidney line MDCK for Influenza Virus; the pig cell line PK15 for PPV. VERO, MARC145, PK15 and MDCK cells were cultured in EMEM added with 10% (growth medium) or 5% (maintenance medium) foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 g/ml); RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 g/ml) was used for JJHAN cells. All the cell lines were incubated at 37C with 5% CO2. The viral strains of HSV-1, ADV-5, COXB5 and MV were clinical isolates, laboratory adapted through serial passages (>50) on VERO cells. The Influenza Virus strain used was the highly neurotropic cell culture adapted WSN33 strain (A/H1N1). For HHV-6, the U1102 strain (variant A) was employed, whereas the two swine viruses tested were reference strains: NADL-2 for PPV and the ATCC strain (Cat. N VR-2402) for PRRSV. Batches of each virus were prepared, titrated on the suitable cell line and kept frozen at -80C until they were used for the experiments. Cytotoxicity Assay The MTT test [8] was used to evaluate the effects of the different concentrations of HA on cell viability. Briefly, serial dilutions of HA from 4 mg/ml to 0.5 mg/ml were prepared in maintenance medium and added (250 l/ well) to 24 hr-old cultures of each line. Each dilution was always tested in triplicate and, in each of the 3 experiments car (...truncated)