Inhibition Effect Of Antimicrobial Peptide Caerin1.1 On The Propagation Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus is an enveloped pathogen with single-stranded positive-sense RNA which causes porcine epidemic diarrhea. The clinical symptoms include acute diarrhea and vomiting in pigs of all ages, the mortality of infected piglets can be as high as 80% to 100%.Veterinarians now use vaccines to halt the transmission of PEDV, however there still has no effective medicines. Antimicrobial peptides(AMPs) are peptides derived from organisms to protect themselves in innate immunity, some AMPs have excellent antiviral activity against the enveloped viruses. In this research the in vitro antiviral activity and machanisms of Caerin1.1 against PEDV was tested, the results showed that Caerin1.1 could directly disrupt the viral memberane and interfere with the virus releasing process.1. Determination of the AMP with the strongest antiviral activity from 5 different AMPs Caerin1.1, Piscidin1, Maculatin1.1, Lactoferricin B and Indolicidin were separately incubated with PEDV for 1h before infecting the cells.The virus titration experiment showed that Caerin1.1 and Piscidin1 are the two most effective against PEDV. They both have low cell cytotoxicity at the concentration of 110μg/m L, the morphology of the cells remained almost normal and the cell viability values were still above 80%.2. The optimization of the most suitable incubation conditions and the inhibit effects Different parameters of incubation temperatures and time lengths were measured, and the results showed that the appropriate condition for incubation of Caerin1.1 and PEDV is 1h in 37℃. Caerin1.1 has remarkable dose-dependent inhibition effects on three different PEDV strains, but the combination of Caerin1.1 and Piscidin1 showed no synergistical inhibiting effects than Caerin1.1 alone.3. Caerin 1.1 could inhibit PEDV replication through directly destroying viral membrane Caerin1.1 was used in three different conditions: a. incubation of Caerin1.1 with cells for 1h before infection; b. Simultaneous incubation of Caerin1.1 and PEDV for 1h before infection; c. administration of Caerin1.1 1h after cell was infected with PEDV. The incubation of Caerin1.1 and PEDV leaded to the decline of virus titers for almost 1000 times, the other two ways also showed slight titer decreases, implying that Caerin1.1 can damage the viral particles and has slight inhibiting effects during the virus multiplication cycle.The results of Transmission electron microscope showed that Caerin1.1can damage the integrity of the viral envelop, thus the infection is suppressed. Westren blot analysis indicated that the expression of PEDV-N protein decreased dramatically in cells infected with pre-treated PEDV.4. The effects of Caerin1.1 on different viral replication stages The attachment and entry stages were extended by incubating the infected cells with Caerin1.1 in 4℃ for 3h, the results revealed that even with a higher dose of Caerin1.1, the trend of the virus titers showed no significant difference comparing with the virus control group, this means that there are no remarkable inhibition effects of Caerin1.1 with PEDV at the attachment and entry stages.Caerin1.1 was inoculated after virus entry, the supernatants and intracellular virus titers were measured seperately, the similar results of the intracellular virus titers between the Caerin1.1 treated group and PEDV control group revealed that Caerin1.1 has no effects on inhibiting the biosynthesis and assembling processes. But the supernatant virus titers in the Caerin1.1 treated group decreased dramatically, the rising trend of the supernatant virus titers was slowed down and it was also dose-dependent, implying that Caerin1.1 could inhibit the release of the newly synthesized PEDV particles.The results of the confocal assay demonstrated that Caerin1.1 and PEDV both localized in the cytoplasm. In control group, Caerin1.1 was in a randomly scattered status, but in PEDV infected cells, Caerin1.1 aggregated at the sites where the viruses gathered.In conclusion, Caerin1.1 and Piscidin1 were screened as effective antimicrobial peptides to halt the infection of PEDV. Caerin1.1 could inhibit the infection of PEDV by damaging the integrity of the viral envelop, and interfering with the virus release process. The results could lay a foundation for antimicrobial peptide application and novel virucide development against PEDV.