Longicin

Background: Longicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat virus (LGTV), a tick-borne flavivirus. Methods: Longicin P1 and P4 peptides were chemically synthesized. Antiviral activity of the longicin peptides against LGTV was evaluated through in vitro virucidal assays, wherein the antiviral efficacy was determined by reduction in number of viral foci and virus yield. Additionally, longicin P4 was also tested for its activity against human adenovirus, a non-enveloped virus. Lastly, to assess the importance of longicin on the innate antiviral immunity of H. longicornis ticks, gene silencing through RNAi was performed. Results: Longicin P4 produced significant viral foci reduction and lower virus yield against LGTV, while longicin P1 failed to demonstrate the same results. Conversely, both longicin partial analogs (P1 and P4) did not show significant antiviral activity when tested on adenovirus. In addition, longicin-silenced ticks showed significantly higher virus titer after 7 days post-infection but a significantly lower titer was detected after an additional 14 days of observation as compared to the Luc dsRNA-injected ticks. Mortality in both groups did not show any significant difference. Conclusion: Our results suggest that longicin P4 has in vitro antiviral activity against LGTV but not against a non-enveloped virus such as adenovirus. Likewise, though most cationic antimicrobial peptides like longicin act directly on target membranes, the exact mechanism of membrane targeting of longicin P4 in enveloped viruses, such as LGTV, requires further investigation. Lastly, while the in vitro virucidal capacity of longicin P4 was confirmed in this study, the role of the endogenous tick longicin in the antiviral defense of H. longicornis against LGTV still remains to be demonstrated.

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.