Johnston

Lake Sturgeon Herpesvirus 2 is Virulent to Juvenile Great Lakes Lake Sturgeon (Acipenser fulvescens) and Susceptible to Multiple Disinfectants in vitro

1,2,*Amber E. Johnston, 1,2Megan A. Shavalier, 2Kim T. Scribner, 3Esteban Soto, 3Susan Yun, 4Andrew D. Winters, 2Douglas L. Larson, 1,2,5Thomas P. Loch

1Michigan State University - Aquatic Animal Health Lab, Aquatic Animal Disease Ecology Program; 2Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University; 3Department of Epidemiology, School of Veterinary Medicine, University of California-Davis; 4Department of Biochemistry, Microbiology, and Immunology, School of Medicine, Wayne State University; 5Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University; *Current Affiliation: United States Department of Agriculture – Agricultural Research Service, Aquatic Animal Health Research Unit, 990 Wire Rd, Office 103C, Auburn AL, 36832

ABSTRACT

Historically, little had been known about infectious diseases affecting Great Lakes lake sturgeon (Acipenser fulvescens; GL-LST), despite dramatic population declines and substantial efforts to restore this iconic species. During a recently completed study, an Alloherpesvirus (Family Alloherpesviridae) was detected and isolated from spawning GL-LST in Michigan (USA). While full taxonomic classification is pending, genomic analyses revealed the virus was distinct from known alloherpesviruses, including an alloherpesvirus recently detected in lake sturgeon in Wisconsin (USA; i.e., Lake Sturgeon Herpesvirus; LSHV). Resultantly, this virus isolated in Michigan (USA) was tentatively named Lake Sturgeon Herpesvirus 2 (LSHV-2). This first isolation of LSHV-2 afforded the opportunity to 1) evaluate fish health risk by determining in vivo virulence under controlled laboratory conditions, and 2) evaluate virus susceptibility to multiple disinfectants currently used in fish culture. To assess the virulence of LSHV-2 in juvenile GL-LST, six replicate groups of eight fish were exposed via immersion bath to a virus solution produced in vitro, while six replicate groups of fish (eight fish/group) were mock exposed to a negative control suspension containing cultured cells with no virus. Results showed that gross signs of disease (e.g., skin ulceration, peri-oral hemorrhaging, lethargy) manifested in virus-exposed replicates as early as 10 days post exposure (DPE) with mortality beginning 19 DPE and continuing until 76 DPE. An average of 33.3% cumulative percent mortality (CPM) in virus-exposed replicates occurred, in contrast to 0% CPM in mock-exposed replicates. LSHV-2 was re-isolated from clinically diseased fish in the virus exposed group, with no viral isolation occurring from the negative control fish. Next, experiments to assess the efficacy of three routinely used hatchery disinfectants (Virkon™-Aquatic, Ovadine®, Perox-Aid®) were conducted in vitro. LSHV-2 was propagated on a white sturgeon (Acipenser transmontanus) x lake sturgeon hybrid cell line, exposed to each disinfectant at two concentrations in duplicate for 1, 10, and 30 minutes, and then inoculated onto fresh cell cultures. After 14 days of incubation, the tissue culture infectious dose50 was calculated for all replicated and the percent reduction in infectious virus was determined relative to non-treated virus control (2.39x105). When LSHV-2 was exposed to Perox-Aid® for 1, 10, and 30 minutes, the percent reduction was 58.66%, 92.32%, 97.13% (500 ppm), and 92.95%, 95.98%, 99.51% (1,000 ppm). When exposed to Ovadine®, the percent reduction was 99.35%, 99.90%, 100% (50 ppm), and 99.42%, 99.90%, 100% (100 ppm). Lastly, when exposed to Virkon™-Aquatic the percent reduction was 100% for all concentrations (1%, 2%) and timepoints. Based upon these results, Virkon™-Aquatic, Ovadine®, and Perox-Aid® all represent potential options to help reduce the transmission potential of the virulent LSHV-2 to juvenile GL-LST under field and hatchery conditions.