| Abstract | Antivirusni učinak antimikrobnih peptida bombinina H2 i H4, porijeklom iz kože žabe (Bombina variegata) testiran je na Herpes simplex virusu-1 (HHV-1), virusu s ovojnicom i dvolančanom DNA, a za usporedbu su korišteni drugi virusi za bolje razumijevanje mehanizma navedenih peptida, uključujući virus influence H1N1 koji je RNA virus s ovojnicom i JC Poliomavirus koji je dsDNA virus bez ovojnice. Potencijalni citotoksični učinak bombinina H2 i H4 ispitan je MTT testom. Na temelju rezultata MTT testa izabrane su koncentracije za pokuse u ovom istraživanju. Antivirusni učinak bombinina H2 i H4 na HHV-1 ispitan je “time-of-addition” testom. Naime, stanice su bile inficirane i tretirane s H2 ili H4 (10 i 20 μg/ml) u različito vrijeme infekcije: prije (PRE), tijekom (D) ili nakon (P) adsorpcije virusa na stanice domaćina ili tijekom i nakon (D +P, dvostruka doza) adsorpcijsku fazu i sljedeća 24 sata. Nadalje, radi provjere potencijalnog izravnog učinka bombinina na virione (virucidno djelovanje), virus je prethodno inkubiran sa svakim peptidom 1 sat na 37 ° C, a zatim su smjese korištene za inficiranje stanica. Iz ovih pokusa stanice su prikupljene i kasnije korištene za Western blot, a supernatanti su kasnije korišteni za standardno ispitivanje plakova. Prethodne studije provedene u našem laboratoriju pokazale su da temporin G (Tg) smanjuje infekciju HHV-1 kada se peptid doda tijekom faze adsorpcije virusa (podaci nisu objavljeni). Kako bi se proučio potencijalni sinergijski učinak primjene bombinina H2 i Tg na životni ciklus HHV-1, stanični monoslojevi inficirani su virusom prethodno inkubiranim bombininima, a također su tretirani s Tg-om 1 sat tijekom adsorpcije virusa na stanice domaćina. Nakon 24 sata, supernatanti su prikupljeni i korišteni za izvođenje standardnog testa na plak. Isti pokus je izravno ponovljen na ploči s 96 jažica radi izvođenja Unutar staničnog-Western blota (ICW). Peptidi su zatim testirani na H1N1 virusu influence A isti način kao i na HHV-1, ali na stanicama A549. Umjesto testa plaka korištena je metoda ICW, a stanice su korištene za Western blot. Kako bi se potvrdila virucidna svojstva bombinina na ovojnici virusa, sljedeći je eksperiment bio usmjeren na ispitivanje bombinina na virus bez ovojnice. U tu svrhu odabran je JC poliomavirus (JCPyV). Stanice SVGp12, bile su zaražene 48 sati virusom prethodno tretiranim bombininima, kako je ranije opisano za virucidne testove HHV-1 i IAV. Kako bi se dobila šira slika i bolji pregled informacija, provedeno je ispitivanje “time-of-addition” na isti način kao na HHV-1. Prikupljeni supernatanti i DNA, ekstrahirana iz stanica, korišteni su za Q-PCR analizu. Bombinin H2 i H4 korišteni su u dozama 10 i 20 μg/mL koje nisu pokazale toksični učinak tijekom MTT testa. Rezultati testa plaka i Western blot pokazuju virucidni učinak bombinina H2 u dozama od 10 μg/ml (p <0,001) i 20 μg/ml (p <0,0001) μg/ml i H4 u dozama od 10 μg/ml (p <0,01) i 20 μg/ml (p <0,001) na HHV-1. Bombinin H2 pokazuje snažniji virucidni učinak od oko 2 log smanjenja u usporedbi s infekcijom koja odgovara 99% inhibiciji replikacije HHV-1. Bombinin H2 dodan izravno na HHV-1 u dozi od 10 μg/ml pokazuje aditivni učinak s peptidom temporinom G u dozi od 50 μg/ml tijekom faze adsorpcije. Rezultati Western blota i ICW-a pokazuju da bombinin H2 ima virucidan učinak na H1N1 virus influence. Rezultati dobiveni Q-PCR-om pokazuju da bombinin H2 smanjuje infekciju JC poliomavirusa za 2-log ako su stanice prethodno tretirane peptidom (pre-tretman). Bombinin H2 pokazuje smanjenje infekcije za 1-log ako se doda u drugim stadijima infekcije ili izravno na polimiovirus JC. Bombinin H4 ne pokazuje antivirusni učinak na JC Poliomavirus. Prema našim saznanjima ovo je prvo izvješće o antivirusnom djelovanju bombinina H2 i H4. |
| Abstract (english) | Antiviral effect of antimicrobial peptides bombinin H2 and H4 originated from frog skin (Bombina variegata) was tested on enveloped, dsDNA, Human aplhaherpesvirus-1 (HHV-1) and for comparison other viruses were used to understand the mechanism of peptides better, including H1N1 influenza virus which is an enveloped RNA virus and JC Polyomavirus which is non-enveloped dsDNA virus. MTT assay was used to test possible cytotoxicity of bombinin H2 and H4 as well as to choose appropriate concentrations for further experiments.. To test antiviral effect of bombinin H2 and H4 on HHV-1 time of addition assays were performed. Cells were infected and treated with H2 or H4 (10 and 20 μg/ml) at different times of infection: before (PRE), during (D) or after (P) the virus adsorption to the host cells or during and after (D+P, double dose) the adsorption phase and for the following 24 h. Moreover, to verify the potentially direct effect of bombinins on virions (a virucidal action), virus was preincubated with each peptide for 1 h at 37°C, and then the mixtures were used to infect the cells. From these experiments cells were collected and later used for Western blot and supernatants later used for plaque assay. Previous studies performed in our lab demonstrated that temporin G (Tg) reduces HHV-1 infection when the peptide is added during the viral adsorption phase (data not published). To study the potential synergic effect of bombinins and Tg administration on HHV-1 life cycle, cellular monolayers were infected with virus preincubated with bombinins and were also treated with Tg for 1 h during the virus adsorption to the host cells. After 24 h, supernatants were collected and used to perform a standard plaque assay. The same experiment was directly repeated in a 96-well plate to perform an Odyssey assay. To conclude are bombinins H2 and H4 virucidal on other viruses than HHV-1 Influenza A virus was tested in the same manner as for HHV-1 but on A549 cells. Instead of plaque assay used method was ICW and cells were used for Western blot. To verify the virucidal properties of bombinins on viral envelope, the next experimentation was addressed to test bombinins against a non-enveloped virus. To this aim, JC polyomavirus (JCPyV) was chosen. SVGp12 cells, were infected for 48 h with bombinins-pretreated virus, as described earlier for HHV-1 and IAV virucidal tests. To get a broad picture, time-of-addition assay was performed as on HHV-1. Collected supernatants and DNA extracted from cells were used for Q-PCR analysis. Bombinin H2 and H4 were used in doses of 10 and 20 μg/mL which do not show toxic effect during MTT assay. Results obtained from plaque assay and Western blot show virucidal effect of bombinin H2 in doses of 10 μg/ml (p<0,001) and 20 μg/ml (p<0,0001) μg/ml and H4 in doses of 10 μg/ml (p<0,01) and 20 μg/ml (p<0,001) on HHV-1. Bombinin H2 shows stronger virucidal effect of about 2-log decrease in comparison to infection which corresponds to 99% inhibition of HHV-1 replication. Bombinin H2 added directly to HHV-1 in dose of 10 μg/ml shows additive effect with temporin G added in the dose of 50 μg/ml during adsorption phase. Results from Western blot and ICW show that bombinin H2 is virucidal on H1N1 influenza virus. Results obtained from Q-PCR show that bombinin H2 reduces infection of JC Polyomavirus for 2-log if cells are pretreated with the peptide. Bombinin H2 shows 1-log reduction of infection if added in other stages of infection or directly to the JC Polyomavirus. Bombinin H4 does not show antiviral effect on JC Polyomavirus. To our knowledge this is a first report on antiviral activity of bombinin H2 and H4. |
