Authored by Ana Maria Viana Pinto*
Abstract
Human alphaherpesvirus 1 is an etiologic agent of infection endemic in the world transmitted by oral to oral oral-genital contact, from mothers causing neonatal herpes and complications such encephalitis or ocular disease. Bovine alphaherpesvirus 5 is an important agent of meningoencephalitis in cattle and has been identified in outbreaks of neurological disease in bovine in several Brazilian States. The aims of this work were to evaluate cytotoxic effect, antiviral properties of the diterpenes 1 and 2 isolated from Canistrocarpus cervicornis. The results of cytotoxic effects in VERO cell showed diterpene 1 more cytotoxic in MTT and Neutral red assay than diterpene 2. The low value to effective concentration required to diterpenes achieve 50% protection to Vero cell showed to diterpene 1 high selectivity index. Both compounds were not cytotoxic to MDBK cell, but they are not able to inhibit Bovine alphaherpesvirus 5 (BoHV-5RJ42/01) replication. Diterpenes 1 and 2 interacted directly with Human alphaherpesvirus 1 (KOS) and BoHV-5RJ42/01 reducing their infectivity on VERO and MDBK cells but they did not inhibit KOS and BoHV- 5RJ42/01 attachment and penetration in Vero and MDBK cells respectively.
Keywords: Canistrocarpus cervicornis; Diterpenes; Human alphaherpesvirus 1; KOS, Bovine alphaherpesvirus 5 BoHV-5; Antiviral
Introduction
Human alphaherpesvirus 1 and Bovine alphaherpesvirus 5 (BoHV-5) are members of the family Herpesviridae, subfamily Alphaherpesvirinae. Human alphaherpesvirus 1 (HHV-1) is including in genus Simplex distributed worldwide [1]. The frequency of seropositive individuals is high, ranging from 50% to over 80% [2]. The Human alphaherpesvirus 1 transmission occurs by direct contact person to person and more likely if blisters or lesions are present. Adolescents and adults frequently get exposed by skin contact but may get exposure by kissing or sexual contact [2]. HHV- 1 infection can occur at different sites of the body. Primary disease is characterized by sore throat, ulcerative and vesicular lesions, gingivostomatitis lymphadenopathy with fever and malaise. Rare, keratoconjunctivitis, encephalitis, and disseminated infections are severe complications of HHV-1 infection [2].
Bovine alphaherpesviruses 5 (BoHV-5) is belonging to the genus Varicellovirus [1], and has been associated with fatal meningoencephalitis in cattle [3] and it has been reported in Australia [4], Argentina [3] and Brazil [5,6] as etiologic agent of neurological disease. BoHV-5 affects mainly but not exclusively young animals. Furthermore, it has been identified from the respiratory [7], genital tracts [8]. BoHV-5 has been described in cryopreserved semen (8) and after experimental infection in oocytes, embryos and inside spermatozoids [9,10].
Bioactive compounds from marine seaweed have been studied to understand their biological effects and application in drugs development. Compounds from marine algae have revealed healthpromoting effects, including anti-oxidative, anti-inflammatory, antimicrobial, anti-cancer and antiviral effects [11]. Canistrocarpus cervicornis (Kutzing) De Paula and De Clerck is important and abundant seaweed of Brazilian coast [12]. From this seaweed was described the isolation of many dolastanes and secodolastanes diterpenes [13-17]. Since then a variety of biological activities of these diterpenes have been published [18-21], including activity against Human alphaherpesvirus 1 [22] and against human immunodeficiency virus 1 [23]. Our purposes in this work were to evaluate the cytotoxicity effect and antiviral activity of two diterpenes purified from crude extract from C. cervicornis and to determine the activities of these compounds on HHV-1 KOS and BoHV-5RJ42/01 replication.
Materials and Methods
Algal collection
Specimens of brown seaweed C. cervicornis (Kützing) De Paula and De Clerck were collected at Praia do Velho in Angra dos Reis, in the south of Rio de Janeiro State, Brazil (lat. 23˚01’S, long. 44˚ 00’W). Voucher specimens (HRJ 10754) were deposited in the Herbarium of the Universidade do Estado do Rio de Janeiro (UERJ). The seaweeds were washed with local sea water and separated from sediments, epiphytes, and other associated organisms.
In order to prepare crude extract air-dried seaweeds were exhaustively extracted with CH2Cl2 at room temperature. The extract was evaporated under reduced pressure, yielding a brownish residue. The crude extract was acetylated as described previously by our group [23]. The acetylated extract was subjected to silica gel chromatography (3 x 40 cm) eluted with 100% n-hexane to 100% EtOAc, in steps of 10% and 100 mL, from which 49 fractions were obtained. The fractionation was monitored by thin layer chromatography and spots of diterpenes were detected as pink-colored spots after heating the plated specimens at 100°C for 3 minutes. The fraction 9 yielded the dolastane diterpene 1. The acetylation reaction and silica gel chromatography were repeated on another aliquot of crude extract, from which 44 fractions were obtained. The fractions 26, 27, and 28 yielded the dolastane diterpene 2. The structures of the diterpenes were assigned by comparison of physical and spectroscopic data with reported values [15] (Figure 1).
Cells and virus and acyclovir
African green monkey kidney VERO-ATCCCCL81 originally obtained from ATCC and Madin-Darbin bovine kidney (MDBK) cell lineage, originally obtained from ATCC (CCL-22) were grown and maintained in Eagle’s minimum essential medium (EMEM) containing 292 μg/mL of L-glutamine, 50 μg/mL of gentamicin and supplemented with 2-5% heated inactivated certified fetal calf serum (Invitrogen). Overlay medium for plaque assay consisted of EMEM 2 x plus in agarose 2% (1:1). Human alphaherpesvirus 1 (HHV-1 KOS) stocks were prepared by infecting subconfluent monolayers of Vero cells at a multiplicity of infection of the 1 MOI. Infected cells were incubated at 37 °C under a 5% CO2 humidified atmosphere and harvested when total cytopathic effect was observed. After three freeze-thaw cycles, cell debris was removed by low-speed centrifugation (2,000 x g at 4°C for 15 min) and titers were determined by plaque assays on Vero cells (24) and expressed as plaque forming units (P.F.U per mL-1). Aliquots of HHV-1 KOS were stored at -70°C until use. Bovine alphaherpesvirus 5 (BoHV- 5RJ42/01) was isolated and characterized previously (Pinto et al. 2015). Virus stocks were propagated in MDBK cells, and the virus titer was determined by plaque assay (PA) (24). The title of the virus was expressed as plaque-forming units (PFU per mL-1). Acyclovir (ACV) used as standard compound purchase from Sigma-Aldrich. All compounds were dissolved in dimetylsulphoxide (DMSO) to obtain stock solutions of 50 mM then diluted in the culture medium without serum to obtain the desired compound’s concentration before use. The final concentration of DMSO was < 0.1%.
Cytotoxicity assay
The cytotoxic effect of seaweed diterpenes 1, 2 and acyclovir was assayed by three methodologies (3-(4,5-dimethylthiazol- 2yl)-2,5diphenyl tetrazolium bromide (MTT) (25), neutral red (NR) (26) and violet crystal ( VC) (27) in Vero and MDBK cell with some modification. Vero and MDBK cells at density of 3x103 were grown in 96 well microplates at 37 °C under a 5% CO2 humidified atmosphere. After 24 hours culture media were replaced with EMEM containing different concentrations of diterpene 1 and 2 and standard compound acyclovir (12.5, 25, 50, 100 and 200 μM) in triplicate. Vero and MDBK cell untreated were used as control. After 72 hours of incubation the supernatants were discarded, and plates separated in three groups for revelation.
the first group of the plates was added MTT (100 μL of 1 mg mL-1 in EMEM) in each well following reincubation for 3h 37 °C under a 5% CO2 humidified atmosphere. Then the medium was discarded and DMSO (100 μL) added before a further incubation for 30 min.
red 0.01% were added in all plates wells and after 3 h at 37 °C under a 5% CO2 humidified atmosphere then supernatants were discarded and cell fixed with formaldehyde [100 μL of 1 mg mL-1 in PBS (NaCl 130 mM; KCl2 mM; Na2HPO4 2H2O6 mM; K2HPO4 1 mM, pH 7.2)] and after reincubation for 15 min the formaldehyde was removed and the neutral red was extracted from cell with 100 μL of the acetic acid1% in methanol 50%.
In the third sets of the plates was added violet crystal (VC) 0.5% in acetic acid 30% (100 μL). The plates were maintained for 30 min at room temperature. Then cells were washed with water and dried at 37ºC following elution of VC by addition of methanol P.A (100 μL). Finally, optical densities of all three experiments were measured at 520 nm in microplate reader and results were expressed as the 50% cytotoxic concentration (CC50). The compound concentration required to reduce the optical density in relation to the cell control was calculated by a linear regression analysis.
Screening of antiviral activity of compounds by plaque reduction assay
An antiviral screening test was performed in Vero and MDBK cells grown in 24 well microplates were inoculated with 200 P.F.U of the HHV-1KOS and BoHV-5RJ42/01 respectively as described previously (24) with some modifications (28). After one hour for virus adsorption at 37 °C with a 5% CO2 atmosphere the inoculum was discarded for addition of the differents concentration of diterpenes 1, 2 and ACV (12.5, 25, 50 and 100 μM). The plates were reincubated for 72 h at 37 °C with a 5% CO2 atmosphere. Then cell monolayer was fixed and stained with crystal violet (1%) in formalin (10%). The antiviral concentration of compounds of 50% effectiveness (EC50) was defined as the concentration required reducing plaque number by 50% in the treated cells compared to untreated ones by the formula:
Percent of inhibition (%) = Number total of control plaques – Number total of tested plaques x 100
Number of total of control plaques
Virus inactivation of infectivity assay
In brief HHV-1KOS and BoHV-5RJ42/01 suspension containing 1x104 P.F.U mL-1 were mixed with 12.5, 25 and 50 μM of compounds and kept at room temperature (24ºC) for 1 h. Meanwhile, control of untreated virus suspension was performed in the same conditions. After that they were diluted, and residual infectivity was determined by plaque assay as described previously [24].
Inhibition of virus attachment assay in pretreated cell and unpretreated cell
Virion attachment assay was performed as described earlier [24] with some modifications [28]. Vero and MDBK cells monolayers grown in 24 well microplates were pre-chilled at 4°C for 1 h. The medium was aspirated and a set of the monolayers were inoculated with 200 P.F.U of HHV-1 KOS or BoHV-5RJ42/01 after pre-treatment with compounds (12.5 and 50 μM) for 1 h at 4ºC. Other set of monolayers previously chilled were inoculated with HHV-1 KOS or BoHV-5RJ42/01 in the presence or absence of differents concentrations of diterpene 1, diterpene 2 and ACV (12.5 and 50 μM). Next, all plates were reincubated for 3 h at 4 °C. Then cell monolayers were washed three times with MEM-E and covered with overlaid medium and reincubated. After 72 h cell monolayers were stained and the attachment-inhibition percentage was calculated as described in the item: Screening of antiviral activity by plaque reduction assay.
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