Detection of Novel Fish Viruses in Wisconsin Sportfish: Implications of Co‐Infections for Disease Emergence

Fishing is a major economic driver in Wisconsin, with approximately 1.4 million licences purchased each year, generating $2.86 billion (USD) for Wisconsin (Fishing Wisconsin 2025). However, recreational angling in Wisconsin faces several threats. Infectious disease is among the most serious of these, along with habitat loss/degradation, pollution and invasive species (Walker and Winton 2010). For example, the introduction of viral hemorrhagic septicemia virus into the Great Lakes ecosystem in 2003 caused numerous fish kills across multiple states (Faisal et al. 2012; EPA 2019). The losses resulting from VHSV highlight the importance of new, emerging infectious diseases to the health and sustainability of Wisconsin's fisheries. Emerging viruses are those new to a population, or increasing rapidly in incidence or geographic range (Burrell et al. 2017) and have contributed to considerable mortality in fish populations, leading to economic losses in aquaculture and threats to wild fish stocks (Langdon and Humphrey 1987; Hanson et al. 2001; Deng et al. 2011; Costa and Holmes 2024). An example is infectious haematopoietic necrosis virus (IHNV), a rhabdovirus which causes haemorrhaging and septicemia in fish. A series of outbreaks occurred in British Columbia and Washington State between 1992 and 2012, resulting in Atlantic salmon mortalities of up to 78%. As a result, many smaller farms went bankrupt and tens of millions of dollars were lost to culling of Atlantic salmon to stop the spread of the virus (Lafferty et al. 2015). IHNV has also been associated with mortality in wild juvenile migratory salmon, highlighting the threat IHNV poses to both wild and captive-raised Salmon (Jeffries et al. 2014). Prevention and prompt detection of pathogens are crucial to reduce societal, ecological and economic costs of fish disease (Asad 2023). For fisheries managers, knowledge of which viruses are cause for concern is essential for informing management practices (Nerland et al. 2021). In a previous study, we surveyed sportfish across the state of Wisconsin and used metagenomics to screen for viruses in serum. Through this approach, we identified 19 viruses from 11 viral families; 17 of which had been previously undescribed (Ford et al. 2024). For the present study, we selected six viruses related to known fish pathogens and used newly developed RT-qPCR assays to investigate their geographic occurrence and true prevalence in Wisconsin sportfish. We analysed 453 blood serum samples from four species of fish collected by the Wisconsin Department of Natural Resources as part of surveys of fish health across Wisconsin: walleye (Sander vitreus; n = 114), bluegill (Lepomis macrochirus; n = 127), brown trout (Salmo trutta; n = 91) and northern pike (Esox lucius; n = 121). Sampling locations consisted of 44 inland waterbody sites across Wisconsin spanning 12 watersheds (n = 13 to 52; Figure S1, Table S1) and fish appeared to be clinically healthy. Samples were collected from 2016 to 2017 as previously described in Ford et al. (2024). We selected viruses from Ford et al. (2024) for RT-qPCR assay development based on the following criteria: (1) they were present in either bluegill, brown trout, northern pike or walleye, (2) they were related to viruses that had been successfully cultured previously (Kawana and Kaneko 1968; Otsuki et al. 1979; Blindheim et al. 2015; Tattiyapong et al. 2017; Wang et al. 2018; Waltzek et al. 2019) and/or (3) were related to viruses, or were themselves known to be associated with disease in fish (Bacharach et al. 2016; Vendramin et al. 2019; Quinteros et al. 2022). These viruses were: eaulepmac virus 1 and mislepmac virus 1 (f. Matonaviridae), lipesoluc virus 1 (f. Amnoonviridae), pelsanvit virus 1 (f. Peribunyaviridae), tursanvit virus 1 (f. Coronaviridae) and piscine orthoreovirus 3 (f. Spinareoviridae). Primers, probes, gBlocks and RNA standards were designed using PrimerQuest Tool (Integrated DNA Technologies, IA, USA) and checked for specificity using Geneious v2025.0.3 (https://www.geneious.com) and BLAST nucleotide similarity search (Altschul et al. 1990; National Center for Biotechnology Information (NCBI) 2025; Table S2). We extracted RNA using the QIAamp UltraSens Virus Kit (Qiagen, MD, USA) as per manufacturer's instructions, with the exception of the final elution volume of 150 μL. Samples underwent RT-qPCR using the GoTaq Probe 1-Step RT-qPCR System (Promega, WI, USA). We ran reactions in 20 μL volumes containing: 10 μL of 2× master mix, 0.4 μL of 50× RT-Enzyme mix, 1 μL each of 10 μM forward and reverse primers, 0.5 μL of 10 μM probe and 4.6 μL of nuclease free water. We placed samples on 0.1 mL MicroAmp Optical 96-Well Reaction Plates (Thermofisher Scientific, MA, USA)/Hard-Shell 384-Well PCR Plates (skirted; Bio-Rad, CA, USA) alongside a no-template control, a negative extraction control, a gBlock standard and an RNA standard. We then sealed plates with PCR Plate Heat Seal (BIO-RAD, CA, USA) and prepared them on an epMotion 5075 (Eppendorf, CT, USA) liquid handling machine, after which we ran PCRs on a CFX Opus 384/96 Real-Time PCR System (Bio-Rad, CA, USA) with the following cycle settings: 45°C for 15 min, 95°C for 2 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. We ran samples in duplicate and considered them positive if a sigmoidal amplification curve was present in both replicates above the fluorescence threshold. Samples in which only one replicate amplified were repeated until both replicates reached a consensus. Positive amplicons were confirmed by the presence of a band of the predicted length (approximately 100 bp) on a 2% agarose gel. From the Wisconsin DNR fish health survey, a total of 14/453 fish tested positive for any one of the six viruses investigated in this study (all controls performed as expected). Bluegill contained the highest diversity of viruses (n = 3), with only a single virus detected in walleye, northern pike or brown trout (Figure 1). Eaulepmac virus 1 and mislepmac virus 1 (f. Matonaviridae) were restricted to bluegill only with a prevalence of 3.1% (4/127) and 7.1% (9/127) respectively. Lipesoluc virus 1, tursanvit virus 1 and piscine orthoreovirus 3 RT-qPCR assays were unable to detect further presence of these viruses in the fish. Pelsanvit virus 1 was the only virus found to be present in multiple fish species, bluegill (0.8%; 1/127) and walleye (0.9%; 1/114), when both species were sampled from the same site. We detected both viruses from the Matonaviridae family in two bluegill. We detected the presence of eaulepmac virus 1 in the Saint Croix, Upper Fox, Upper Wisconsin and Lower Chippewa watersheds (Figure 1). We found Mislepmac virus 1 in the Saint Croix, Upper Fox and Wolf River watersheds and pelsanvit virus 1 in the Upper Wisconsin watershed only. Watersheds with multiple viruses present included; Lower Chippewa, Saint Croix, Upper Fox and Upper Wisconsin. Previously we identified 19 viruses from 103 blood serum samples taken from five freshwater species of fish collected during a state-wide survey of Wisconsin from 2016 to 2020 (Ford et al. 2024). In this current study, we further assessed the prevalence and geographic distribution of six of these viruses using novel RT-qPCR assays run on archived serum samples from across Wisconsin. The most prevalent viruses in this study were both from the Matonaviridae family, eaulepmac virus 1 and mislepmac virus 1, spanning 6 and 4 watersheds respectively, while pelsanvit virus 1 remained contained to the Upper Wisconsin River watershed, as previously described (Ford et al. 2024). We found multiple viruses in several watersheds. We detected both matonaviruses in the Saint Croix, Lower Chippewa and Upper Fox watersheds, and we detected eaulepmac virus 1 and pelsanvit virus 1 (f. Peribunyaviridae) in the Upper Wisconsin watershed. Viral co-infections can have significant effects on virus evolution and are certainly a threat to fish health (Wu et al. 2024). For example, co-infections are prerequisite for recombination between viruses, which can result in increased transmissibility or pathogenicity (Du et al. 2022). The detection of two matonaviruses within the same watersheds, and in some cases, within the same individual bluegill, highlights the potential for the emergence of recombinant viruses in these environments arising from co-infections. To date, only two viruses from the Peribunyaviridae family have been detected in fish, both of which were first identified in Wisconsin. Largemouth bass bunyavirus was detected in largemouth bass during a routine health assessment in 2009; however, its pathogenesis and host range are currently unknown (Waltzek et al. 2019). Pelsanvit virus 1, first described in our previous study (Ford et al. 2024) was detected in walleye. In this current study, we detected pelsanvit virus 1 RNA in bluegill, suggesting a possible extension of its host range. Both of our studies found this virus in the Upper Wisconsin River watershed, unlike the largemouth bass bunyavirus (Pool 10 of the upper Mississippi River) suggesting peribunyaviruses in fish are more prevalent in Wisconsin than previously thought. However, we do recognise that any conclusions made in this study are limited by the detection ability of the qPCR assay. The detection rate of viruses in this study was low overall, though this is not uncommon in pathogen surveillance, but may be due to the large extraction elution volume needed for this study or levels of viremia below detectable levels. All fish sampled were apparently healthy at the time of sampling. The clinical effects of these viruses are therefore unclear. However, viruses in the families we have identified can be cultured, and such viruses often grow in cell cultures during investigations of fish kills or in routine health screening. Knowing the prevalence and distribution of these viruses in healthy fish will help determine whether future detections using culture or molecular methods are causally related to morbidity/mortality or whether they are incidental findings. Future studies that isolate these viruses through culture will enable characterisation of their genomes and in vivo experiments, to determine their pathogenicity and transmissibility. T.L.G. and E.M.L. designed the study; W.A.T. collected the samples; C.E.F., I.S. and K.V.R. processed the samples; C.E.F. analysed the data and prepared the manuscript with edits from all co-authors. All authors have read and agreed to the published version of the manuscript. The authors would like to thank the WI DNR Bureau of Fisheries Management for their efforts in the collection of samples used in this project and Nikolas Grueneis for his input in the planning stages. We would like to thank the reviewers for taking the necessary time and effort to review the manuscript. Usage of trade names does not imply endorsement by the US Government. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service. The samples used in the study were archived samples previously collected by the Wisconsin Department of Natural Resources for health surveys. The authors declare no conflicts of interest. All data can be accessed from the Supporting Information. Figure S1. Map of sampling locations throughout Wisconsin where L. macrochirus (bluegill), S. trutta (brown trout), E. lucius (northern pike) and S. vitreus (walleye) were collected in this study. Table S2. Primer and probe sequences for the viruses investigated in this study. Table S1. Sampling locations and RT-qPCR results for the four species of fish sampled across Wisconsin from 2016 to 2017. 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