title: Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis virus surrogate Langat virus.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. melbourne.talactac@gmail.com.|Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. melbourne.talactac@gmail.com.|Department of Clinical and Population Health, College of Veterinary Medicine and Biomedical Sciences, Cavite State University, Cavite, 4122, Philippines. melbourne.talactac@gmail.com.
authors.forename: Melbourne Rio
authors.initials: MR
authors.lastname: Talactac
authors.affiliation: Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku kita-18 nishi-9, Sapporo, Hokkaido, 060-0818, Japan. kyoshii@vetmed.hokudai.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yoshii
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. vet_hiro_kagoshima@yahoo.co.jp.|Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. vet_hiro_kagoshima@yahoo.co.jp.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Maeda
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. seigi26jp@yahoo.co.jp.|Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. seigi26jp@yahoo.co.jp.
authors.forename: Kodai
authors.initials: K
authors.lastname: Kusakisako
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. jhunemman@yahoo.com.
authors.forename: Emmanuel Pacia
authors.initials: EP
authors.lastname: Hernandez
authors.affiliation: Department of Parasitology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami, Sagamihara, Kanagawa, 252-0374, Japan. tsujin@med.kitasato-u.ac.jp.
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Tsuji
authors.affiliation: Zen-noh Institute of Animal Health, Ohja, Sakura, Chiba, 285-0043, Japan. acarikf@nifty.com.
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.affiliation: Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, Los Baños, Laguna, 4031, Philippines. rlgalay.dvm@gmail.com.
authors.forename: Remil Linggatong
authors.initials: RL
authors.lastname: Galay
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. tetsuya@ms.kagoshima-u.ac.jp.|Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. tetsuya@ms.kagoshima-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, 753-8515, Japan. masamimochizuki@gmail.com.|Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan. masamimochizuki@gmail.com.
authors.forename: Masami
authors.initials: M
authors.lastname: Mochizuki
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000260:Adenoviruses, Human / Q000187:drug effects / Q000502:physiology; D000818:Animals; D004669:Encephalitis Viruses, Tick-Borne / Q000187:drug effects* / Q000502:physiology; D026863:Ixodidae / Q000737:chemistry*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects
chemical_list: C096944:longicin; D015107:4-Butyrolactone
references.citation: Cell Host Microbe. 2008 Jan 17;3(1):11-9
references.pmid: 18191790
references.citation: MBio. 2015;6(3):e00614
references.pmid: 26045539
references.citation: Front Cell Infect Microbiol. 2013;3:26
references.pmid: 23875177
references.citation: Biotechnol Lett. 2005 Sep;27(18):1337-47
references.pmid: 16215847
references.citation: Peptides. 2006 Apr;27(4):931-40
references.pmid: 16226346
references.citation: Adv Virus Res. 2003;60:233-72
references.pmid: 14689696
references.citation: Cancers (Basel). 2013 Nov 01;5(4):1413-25
references.pmid: 24202451
references.citation: Dev Comp Immunol. 1999 Jun-Jul;23(4-5):329-44
references.pmid: 10426426
references.citation: Peptides. 2012 Mar;34(1):242-50
references.pmid: 21849158
references.citation: Nature. 2002 Jan 24;415(6870):389-95
references.pmid: 11807545
references.citation: Mol Immunol. 2008 Sep;45(15):3909-16
references.pmid: 18657321
references.citation: Trends Microbiol. 2000 Sep;8(9):402-10
references.pmid: 10989307
references.citation: Parasit Vectors. 2014;7:554
references.pmid: 25443032
references.citation: Virology. 2009 Aug 1;390(2):307-14
references.pmid: 19540545
references.citation: J Exp Biol. 2013 May 15;216(Pt 10):1905-15
references.pmid: 23393286
references.citation: Exp Appl Acarol. 2002;28(1-4):135-40
references.pmid: 14570123
references.citation: Cell Mol Life Sci. 2011 Jul;68(13):2161-76
references.pmid: 21573784
references.citation: Front Biosci. 2008;13:6938-46
references.pmid: 18508706
references.citation: Virol J. 2012;9:6
references.pmid: 22225618
references.citation: Parasitology. 2004;129 Suppl:S3-14
references.pmid: 15938502
references.citation: J Vet Med Sci. 2010 Feb;72(2):149-56
references.pmid: 19940394
references.citation: Biochem Biophys Res Commun. 2009 Sep 18;387(2):393-8
references.pmid: 19615342
references.citation: Lancet. 2008 May 31;371(9627):1861-71
references.pmid: 18514730
references.citation: J Virol. 2014 Nov;88(21):12202-12
references.pmid: 25122777
references.citation: Int J Antimicrob Agents. 2004 Dec;24(6):536-47
references.pmid: 15555874
references.citation: Dev Comp Immunol. 2016 Apr;57:38-47
references.pmid: 26724379
references.citation: BMC Complement Altern Med. 2012;12:214
references.pmid: 23140177
references.citation: Nat Chem Biol. 2013 Dec;9(12):761-8
references.pmid: 24231617
references.citation: Science. 1999 Oct 15;286(5439):498-502
references.pmid: 10521339
references.citation: Infect Immun. 2007 Jul;75(7):3633-40
references.pmid: 17485458