Antiviral Substances from Microalgae Applied in Aquacultures

Although koi herpesvirus (KHV) or scientific Cyprinid herpesvirus 3 (CyHV-3) has a history of causing carp farmers sever economic losses there are still no drugs available against it on the market. Thus, the aim of this study was to find novel substances from microalgae showing antiviral activity against this virus by monitoring infection inhibition and cytotoxic effects in common carp brain (CCB) cells and test further the prophylactic and methaphylactic or therapeutic effects of selected substances in an animal experiment in carp (carried out by Dr. habil. Sven Bergmann, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Germany, 2014). In order to reach these aims, at first, native fresh water and marine microalgae from different locations in South Korea were isolated, identified, using morphological and genotyping methods, and screened for their antiviral properties. Among 80 tested EPS isolates, the isolates of 7 algae species showed antiviral activity against KHV (isolated from diseased fish in Poland, Dr. Magdalena Stachnik, Department of Fish Diseases, National Veterinary Research Institute, 2013) at a multiplicity of infection (MOI) of 0.45 after 16 - 20 days post infection (d p.i.) with no cytotoxicity towards CCB cells. In contrast, commercial antiviral drug inhibiting DNA-translation, -replication or -polymerisation, such as Ganciclovir (Ganc), Cytosine β-D-arabinofuranoside (Ara-C) and Cycloheximide (CHX), applied at 25 μgml−1, showed growth inhibitory or cytotoxic effects with CCB cells and no antiviral activity at the same MOI indicated by expression of different immediate early (IE), early (E) and late (L) KHV life cycle genes (ORF 3L/R, ORF 55, ORF 56, ORF 92 and ORF 114) 16 d p.i.. Additionally, Arthrospira platensis (A. platensis) (NIES 39), well known for the antiviral activity of intra- and extracellular compounds towards e.g. human pytomegalovirus,was investigated. Of all different extracts/isolates from this species the highest antiviral activity with no cytotoxic effects were observed for the dialyzed fraction (MWCO:12 - 14 kDa) of the exopolysaccharides (EPS). However, EPS exhibited inhibitory impact on the growth of CCB cells with a 50% inhibitory concentration (IC50,CCB) at 59μgml−1. Amazingly, it was found in a RT-PCR that EPS, applied at a concentration of >18 μgml−1 and a MOI of 0.45, avoided the KHV replication in CCB cells even after 22 d p.i. entirely. The virus 50% inhibiting concentration (IC50,KHV) of EPS was determined to be <2.1 μgml−1 for a MOI of <0.45 after 12 d p.i.. After completed in vitro studies, 8 different groups of carp (n=50) were treated with crude biomass (BM) or EPS to examine either prophylactic or therapeutic effects after application and infection with KHV (isolated from diseased fish in UK, Dr. Keith Way, The Centre for Environment, Fisheries and Aquaculture Science, 2009) at 104−6 TCID50 ml−1 in the pond water. BM was applied orally and solved in the water at concentration of 1.28 g d−1 and aquarium (400 L). EPS was dissolved in aquarium water at an always constant concentration of 32mgL−1 of EPS in 400 L. After the experiment carp were examined for the presence of clinical (morbidity) as well as for the losses (mortality) and also were tested serologically. Result showed that BM, applied metaphylactic, hardly helps against KHVD. However, a significant reduced mortality was found in the EPS group (prophylaxis) only 5.7% cumulative mortality and amazingly also in the therapeutic EPS group with 7.5 %, in comparison to the positive control group (36%). Moreover, all carp survived of infected control groups showed in terms of morbidity a severe koi herpesvirus disease (KHVD), while much better results were found in survivors of pro- and metaphylactic EPS group. Those groups did not displayed any sign of KHVD or only 12.2 %, respectively. Additionally, both EPS groups showed 14 - 20.4% external but healed signs of the disease. Serological result showed that metaphylactic EPS group developed weak antibodies against KHV (60) but with a very good surviving rate after EPS application. Whereas, prophylactic EPS group developed much higher reciprocal KHV antibody titres (420) against KHV with more sera positive for KHV antibodies (70%), with the highest surviving rate after EPS application. To sum it up, collected screening results showed to be very promising in respect to finding novel compounds of microalgal origin that can prevent and/or reduce fish infection by KHV. It was demonstrated the first notable cure of KHV infection in carp induced by EPS by a so far unknown immune and/or protection reaction with positive effects on the survival rate after KHVD. Nevertheless, further studies are needed in order to prove EPS effectivity for application in animals.