Study On The Functions And Mechanisms Of Tick Defensins Against African Swine Fever Virus

African swine fever(ASF)is a legally reported animal disease by the World Organization for Animal Health(OIE)and has also been listed as a priority exotic animal disease for surveillance in China.The mortality rate of acute and subacute manifestations of African swine fever is approximately 100%.Since the first outbreak of African swine fever in August 2018 in the Shenbei New District of Shenyang,Liaoning Province,China,the disease has rapidly spread throughout most of the Chinese provinces,causing serious economic losses in the country,and there is no effective medicine or vaccine available.To effectively contain African swine fever,the development of effective vaccines and drugs is paramount,but it is also necessary to analyze its transmission mechanism through the natural vector ticks.In this study,African swine fever virus pS273R protease was successfully expressed by the pET expression system in E.coli.After separation and purification by Ni-chelating affinity chromatography,rTEV protease cleavage,Sephadex G-50 gel chromatography,resource S anion exchange chromatography,and reversed-phase high-performance liquid chromatography,we obtained purified recombinant African swine fever virus pS273R protease.African swine fever virus is the only double-stranded DNA virus transmitted by ticks.Here,we obtained the protein sequences of tick defensins from the GenBank database based on previous research.We compared the protein sequences of a defensin named OPTX-1,which is from the natural vector of African swine fever,Argasidae ticks,and five reported defensins,Persulcatusin,Longicin,Scapularisin,Varisin A1 and Microplusin,which are from five species of Ixodes ticks,that are not capable of spreading African swine fever virus.Then,we refolded linear peptides of five tick defensins of Ixodes and obtained the correctly folded peptides after reversed-phase high-performance liquid chromatography separation and purification.Then,we tested the antiviral activities of these tick defensins against the pS273R protease on an automatic microplate reader after obtaining these samples.The results showed that the Ki values for the inhibition of pS273R protease by tick defensin OPTX-1 from Argasidae were 0.821±0.526 μM,and the Ki values for the inhibition of pS273R protease by five tick defensins from Ixodidae were as follows:the Ki value of Persulcatusin was 12.690±0.438 nM;the Ki value of Scapularisin was 15.765±0.233 nM;The Ki value of Longicin was 20.310±0.127 nM;the Ki value of Varisin A1 was 32.335± 1.888 nM;and the Ki value of the positive drug Bromocriptine for this enzyme was 72.742±12.5 nM.We tested the cytotoxicity of OPTX-1,Persulcatusin,Longicin,Scapularisin,Varisin Al,and Microplusin in Vero cells and PAM cells.The results showed that the survival rates of PAM cells treated with defensin OPTX-1,Persulcatusin,Longicin,Scapularisin,Varisin A1 and Microplusin at a concentration of 100 μM were(54.87±5.80)%、(61.81 ±27.18)%、(79.27±10.16)%、(54.65 ± 15.32)%、(38.16±6.71)%and(53.49 ±7.66)%,respectively and survival rates of Vero cells treated with defensin OPTX-1,Persulcatusin,Longicin,Scapularisin,Varisin A1 and Microplusin at a concentration of 100 μM were(81.28±3.59)%,(51.89±9.41)%,(89.10±3.23)%,(84.36± 14.28)%,(44.79±6.02)%and(56.51±13.83)%respectively.The results show that the cytotoxicity of tick defensins is low,and these tick defensins are potential drug precursors for the treatment of African swine fever.We used Modeller v9.18 software to construct models of three tick defensins,OPTX-1,Persulcatusin,and Longicin,by homology modeling and then used ZDOCK 3.0.2 software to perform molecular docking between the tick defensins and the pS273R protease of African swine fever virus.The results show that tick defensins bind to the active pocket of the pS273R protease and that Longicin,Varisin A1,and Microplusin act on the C232-H168-N187 catalytic triad of the pS273R protein directly.Finally,we used an in vitro infection model to preliminarily evaluate the antiviral function of tick defensins against the African swine fever virus.We studied the antiviral activity of each tick defensin against the African swine fever virus in vitro,and the results showed that the IC50 value of OPTX-1 for ASFV inhibition was 2.99 μM,and that the IC50 value of Persulcatusin for ASFV inhibition was 2.57 μM.The IC50 value of Scapularisin for ASFV inhibition was 2.22 μM.This suggests that tick defensins have potential application value in the treatment of African swine fever.This work discovered the inhibitory effect of tick defensins on the African swine fever virus pS273R protease for the first time and initially evaluated their function in inhibiting African swine fever virus.This work will help us understand the mechanism of the natural vector,Ornithodorus ticks,carrying and spreading African swine fever virus,and it is expected to provide peptide precursor molecules for the development of therapeutic drugs for African swine fever.