A Seasonal Study of Koi Herpesvirus and Koi Sleepy Disease Outbreaks in the United Kingdom in 2018 Using a Pond-Side Test

SIMPLE SUMMARY: Cyprinid herpesvirus (CyHV)-3 and carp edema virus (CEV), the causative agents of koi herpesvirus disease and koi sleepy disease, respectively, are emerging DNA viruses infecting koi and common carp. Similarities in their clinical presentation present difficulties for its on-site identification based on gross pathology. Fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for detecting CyHV-3 and CEV DNA were designed to use border inspection posts and local testing by national authorities for outbreak control. The limit of these tests’ detection (10(2) and 10(3) viral copies for CyHV-3 and CEV, respectively) allows for the amplification of viral DNA in clinical samples in less than 20 min. The assays’ field performance was tested with 63 common carp mucus swabs taken during disease investigations in 2018, and the results validated with the reference laboratory analysis. Overall, the good performance, ease of use, and cost-effectiveness of these tests make them good candidates for a point of care test. However, further work is required to incorporate reliable internal controls and improve the sensitivity of these tests’ asymptomatic testing. ABSTRACT: Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. The limit of detection was 10(2) and 10(3) viral copies under 25 min for CyHV-3 and CEV, respectively. The specificity of the CyHV-3 LAMP assay was 95.6% of 72 fish herpesviruses tested. Sixty-three non-lethal common carp mucus swabs were collected across 16 sites during disease investigations. DNA extractions were performed in under 10 min using the QuickExtract™ digestion buffer. The LAMP amplification of CyHV-3 DNA in mucus swabs from clinical cases was detected from 4 to 13 min in 13 sites, while a co-infection of CyHV-3 and CEV was confirmed by LAMP in a single site. The LAMP results agreed with the results of the reference laboratory. The common carp ef1a was amplified only in 61% of the mucus swabs collected, preventing its use as a robust internal control to distinguish false negatives from invalid tests. After further optimization, these tests could be implemented for border inspection posts surveillance and decentralizing testing during disease outbreaks.