1Eva M. Quijano Cardé, 1Zeinab Yazdi, 1Susan Yun, 1Ruixue Hu, 2Heather Knych, 1Esteban Soto
1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California - Davis; 2K.L. Maddy Equine Analytical Pharmacology Laboratory, School of Veterinary Medicine, University of California - DavisCyprinid Herpesvirus 3, also known as Koi Herpesvirus (KHV), is a re-emergent virus that affects koi and common carp fish (Cyprinus carpio) and is reportable to the World Organization for Animal Health. KHV disease typically presents as an acute systemic infection, particularly targeting the gills and kidneys, resulting in mortalities as high as 80-100%. KHV survivors can become latent carriers of the virus, shed the virus and develop clinical signs under conditions of stress and permissible temperatures. Currently, there are no vaccines or treatments available in the United States against KHV to reduce morbidity or mortality. The nucleoside analog acyclovir has been shown to decrease the viral load, cytopathic effects, and KHV gene expression in fish cells treated 2h post-infection using an in vitro model. This study aimed to evaluate the pharmacokinetics (PK) of a single acyclovir intracoelomic (IC) injection in koi fingerlings (koi) and to assess its efficacy in decreasing mortality during a KHV mortality event in an in vivo challenge model. Initially, the acyclovir cytotoxicity on the koi fin cell line (KF1) was evaluated by exposing the cells to various acyclovir concentrations for 24h and measuring the release of lactate dehydrogenase. Results suggest that acyclovir at the tested concentrations was non-cytotoxic to KF1 cells when compared to the negative control. Secondly, a single 10 mg/kg acyclovir IC dose was administered to koi and blood was collected from the caudal vein at different time-points after drug administration. Plasma samples were then analyzed via liquid chromatography tandem mass spectrometry. Results indicated an acyclovir peak plasma concentration of 141 𝜇M in 45 min and a half-life of 14h. Additionally, histopathology of various tissues of the acyclovir-treated koi detected no lesions associated with tissue toxicity when compared to untreated koi. Finally, a cohabitation challenge model was performed by exposing subgroups of koi to either a 0.6 KHV pfu/mL bath or a sterile cell culture media bath for 6h. The koi were monitored until mortalities were detected and infection confirmed via qPCR. Then, a single 10 mg/kg IC acyclovir dose or a sterile PBS solution IC injection of similar volume was administered to koi in the treatment or control groups respectively. Mortality was monitored for 30d after drug administration. A statistically significant reduction in cumulative mortality (days 25-30) was detected in the acyclovir-treated group when compared with the positive control group. In addition, a non-statistically significant 3d delay in mortality was observed in the acyclovir group compared to the positive control group. In summary, these results suggest that acyclovir is non-toxic to cells and tissues, and effective in decreasing cumulative mortality during a KHV outbreak in koi fingerlings when administered via a single 10 mg/kg IC injection. In addition, considering the reported acyclovir EC50 in human herpesviruses and the acyclovir PK data in koi obtained in this study, we can hypothesize that a multi-dose protocol could be even more effective in decreasing mortality during an outbreak.