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Vet World
2018 Jun 01;116:824-829. doi: 10.14202/vetworld.2018.824-829.
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Antiviral activity of Acanthaster planci phospholipase A2 against human immunodeficiency virus.
Wijanarko A
,
Lischer K
,
Hermansyah H
,
Pratami DK
,
Sahlan M
.
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Aim: Investigation of antiviral activity of Acanthaster planci phospholipase A2 (AP-PLA2) from moluccas to human immunodeficiency virus (HIV).
Materials and Methods: Crude venom (CV) and F20 (PLA2 with 20% fractioned by ammonium sulfate) as a sample of PLA 2 obtained from A. planci''s extract were used. Enzymatic activity of PLA2 was determined using the degradation of phosphatidylcholine (PC). Activity test was performed using in vitro method using coculture of phytohemagglutinin-stimulated peripheral blood mononuclear cell (PBMC) from a blood donor and PBMC from HIV patient. Toxicity test of AP-PLA2 was done using lethal concentration required to kill 50% of the population (LC50).
Results: AP-PLA2 F20 had activity and purity by 15.66 times bigger than CV. The test showed that the LC50 of AP-PLA2 is 1.638 mg/ml. Antiviral analysis of AP-PLA2 in vitro showed the inhibition of HIV infection to PBMC. HIV culture with AP-PLA2 and without AP-PLA2 has shown the number of infected PBMC (0.299±0.212% and 9.718±0.802%). Subsequently, RNA amplification of HIV using reverse transcriptase-polymerase chain reaction resulted in the decrease of band intensity in gag gene of HIV.
Conclusion: This research suggests that AP-PLA2 has the potential to develop as an antiviral agent because in vitro experiment showed its ability to decrease HIV infection in PBMC and the number of HIV ribonucleic acid in culture.
Figure-1. Peripheral blood mononuclear cell treated with both PLA2 of crude venom (CV) and PLA2 with 20% fractioned by ammonium sulfate (F20) shows decreasing absorbance promptly. The final absorbance is control 0.709±0.02 (n=3), CV 0.67±0.02 (n=3), and F20 0.39±0.05 (n=3).
Figure-2. Toxicity test result of PLA2 to peripheral blood mononuclear cell (PBMC) culture. Addition of PLA2 in PBMC culture significantly decreases total of cell. Final total of cell (day 14) is in control 630,000±31,500 (n=3), crude venom 43,750±21,875 (n=3), PLA2 with 20% fractioned by ammonium sulfate (F20) 1 mg/ml 455,000±45500 (n=3), F20 2 mg/ml 186,250±37,250 (n=3), F20 3 mg/ml 10,000±1,500 (n=3), F20 4 mg/ml 7,500±3,750 (n=3), and F20 5 mg/ml 3750±3000 (n=3).
Figure-3. Gel electrophoresis data after reverse transcriptase-polymerase chain reaction human immunodeficiency virus (HIV) ribonucleic acid (RNA) fluorescence. Lane 1: HIV culture was added to PLA2 (PLA2+HIV). Lane 2: HIV culture without treatment (HIV). Lane 3: Cultured line control of a positive HIV RNA (K+). Lane 4: The culture of peripheral blood mononuclear cell. Lane 5: Cultured line control of a negative HIV RNA (K-). Lane 6: Standard protein.
Figure-4. Cytometry of immunofluorescence assay using fluorescence isothiocyanate in (a) peripheral blood mononuclear cell culture as control negative, (b) no treatment human immunodeficiency virus (HIV) culture as a positive control, (c) HIV culture treat by the addition of PLA2 (PLA2+HIV). The peak by green color on the right side of a threshold shows among of expression of HIV protein. The graph indicates that amount of expressed HIV protein in PLA2+HIV culture is less than no treatment HIV culture.
Figure-5. Percentage of human immunodeficiency virus (HIV) infection in peripheral blood mononuclear cell (PBMC) culture line. After addition of PLA2, proportion of HIV infection was decreased significantly from HIV infection without treatment (HIV) about 9.718±0.802% (n=4) to HIV infection with treatment (PLA2+HIV) 0.299±0.212% (n=4). Meanwhile, control was about 2.15±0.802% (n=4).
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