Ivermectin blocks the nuclear location signal of parvoviruses in crayfish, Cherax quadricarinatus Kim Y. Nguyen a , Kitikarn Sakuna a,b , Robert Kinobe c , Leigh Owens a, ⁎ a Microbiology and Immunology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville 4811, Queensland, Australia b Faculty of Veterinary Science, Rajamangala University of Technology, Srivijaya 80240, Thailand c Physiology and Pharmacology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville 4811, Queensland, Australia abstract article info Article history: Received 23 October 2013 Received in revised form 15 November 2013 Accepted 18 November 2013 Available online 27 November 2013 Keywords: Ivermectin Nuclear location signal Parvoviruses Crayfish Cherax quadricarinatus Parvoviruses have been responsible for major problems in the shrimp aquaculture for decades with few options for control apart from avoidance. As intranuclear viruses for some of their replication, parvoviruses need to use the cell's nuclear transport signals for entry into the nucleus. This study was conducted to see if ivermectin which has recently been shown to block importins in vitro would do so against two presumptive parvoviruses in a freshwater crayfish, Cherax quadricarinatus, model. Crayfish were shown to tolerate ivermectin at 7 μg/kg injected intramuscularly and survival appeared to be enhanced with increasing dose (P ≤ 0.1). Ivermectin dra- matically decreased hypertrophied nuclei caused by presumptive gill parvovirus by ~68% (P ≤ 0.001) after 2 doses of 7 μg/kg reducing from 1591 to 505 affected cells in the gills. The reduction did not increase further with increasing doses. Also, ivermectin appeared to increase the survival of crayfish when challenged with C. quadricarinatus parvo-like virus (CqPV) to levels statistically equivalent to non-infected crayfish but did not ap- pear to affect the number of viral infected cells. There was a negative correlation between the size of crayfish and their longevity (P ≤ 0.05, R 2 = 0.15) with smaller crayfish dying faster when challenged with CqPV. This is the first in vivo testing of ivermectin against viruses and showed that ivermectins do dramatically block some parvo- viruses, possibly by interactions with cellular importins. There may be a therapeutic role for ivermectins in viral reduction in broodstock in crustacean aquaculture. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The penaeid parvoviruses Penaeus monodon Densovirus (PmonDNV, colloquially known as HPV) and Penaeus stylirostris Brevidensovirus (PstBNV, colloquially known as IHHNV) cause many disease issues in penaeids (see reviews Safeena et al., 2010 and Rai et al., 2012). Indeed parvoviruses cause major diseases in many animals including humans, dogs, cats, mink, pigs, cattle, crustaceans and insects. Parvoviruses are intranuclear in their replication and they need rapidly dividing cells in the S-phase to access the cellular DNA replication enzymes. Thus the parvovirus needs to transport their proteins into the nucleus using the cell's nuclear importing molecules, karyopherin also called importin, IMPα/β linked to their nuclear location sequences or signals (NLS). Recently, Owens (2013) identified many possible signals in these penaeid parvoviruses and indeed this current study was spawned from that analysis. Recently, ivermectin and mifepristone were reported to have potent antiviral activity in vitro (Wagstaff et al., 2011, 2012) by preventing ac- tive nuclear transport of the integrase molecule of human immunodefi- ciency virus (HIV)-1. Mifepristone is a specific inhibitor of the nuclear import of the protein integrase, but ivermectin appears to act on IMPα/β-mediated nuclear import generally. This raises the intriguing possibility that ivermectin could be an anti-parvoviral agent if parvovi- ruses use IMPα/β to transit into the nucleus. Ivermectin is an effective antiparasiticide used widely on animal farms including aquaculture against parasites such as sea lice Lepeophtheirus salmonis and Caligus elongatus (Davies and Rodger, 2000) and metacercariae of Clinostomum marginatum (Lorio, 1989). Crustaceans are very sensitive to ivermectin. Loss of action potential in the neuron, loss of motor function and eventual paralysis from avermectin in the brine shrimp Artemia salina, which contains neuro- transmitter gamma-aminobutyric acid (GABA) receptors (Calcott and Fatig, 1984), have been documented. The mysid shrimp, Mysidopsis bahia, was sensitive at 96 h LC 50 0.022 μg/l (Wislocki et al., 1989), whilst the no-observed effect concentration (NOEC) was 4 ng/l , but the 96 h LC 50 for pink shrimp Penaeus duorarum was 1.6 μg/l. The mysid, Neomysis integer showed a 96 h LC 50 of 70 (44–96, 95% CI) ng/l, when immersed (Davies et al., 1997). Through the digestive tract of shrimp Crangon septemspinosa, ivermectin was toxic but not via the gills (Burridge and Haya, 1993). Shrimp could tolerate ivermectin in water at the maximum concentration 21.5 μg/l, but ivermectin was lethal at 96 h LC 50 = 8.5 μg ivermectin/g of food. The shrimp's average weight was 2.76 g, and the feeding rate was 1% body weight per day. The Aquaculture 420–421 (2014) 288–294 ⁎ Corresponding author. E-mail address: leigh.owens@jcu.edu.au (L. Owens). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.11.022 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online