First Isolation of a Herpesvirus (Family Alloherpesviridae) from Great Lakes Lake Sturgeon (Acipenser fulvescens)

Simple Summary Throughout the Great Lakes basin, infectious diseases likewise threaten wild and hatchery reared fishes, and often require management attention. The lake sturgeon (Acipenser fulvescens) is the only sturgeon species native to the Great Lakes, where it is the largest and longest living fish therein. Due to multiple known and unknown factors, current Great Lakes lake sturgeon populations are estimated to be <1% of historical abundances. One potential contributing factor about which little remains known, especially when compared to other Great Lakes fishes, is the impact of infectious diseases. To address this knowledge gap, a two-year disease surveillance study was undertaken, resulting in the detection and isolation of a herpesvirus from lesions observed on wild adult lake sturgeon in two Great Lakes watersheds (Erie and Huron). Genomic analyses revealed the recovered virus was most similar to, yet molecularly distinct from, a herpesvirus recently recovered from lake sturgeon in the Lake Michigan watershed (Wisconsin, USA). This newly described virus, proposed as Lake Sturgeon Herpesvirus 2, proved virulent to juvenile (<1 year old) lake sturgeon, whereby disease and mortality occurred in virus-exposed fish under laboratory conditions. Overall, results from this study highlight the potential threat this newly described virus poses to Great Lakes lake sturgeon conservation efforts. Abstract The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers’ postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.


Introduction
The lake sturgeon (Acipenser fulvescens; Family Acipenseridae) is the only indigenous sturgeon species in the Great Lakes Basin of North America, where it is also the largest and longest living fish [1]. Unique life history traits, such as delayed maturation, protracted spawning, periodicity and low natural recruitment, have placed lake sturgeon at increased risk for human-induced population declines [2]. Indeed, several anthropogenic factors (e.g., overharvesting, habitat degradation, dam construction) have been linked to declines in both numerical abundance and distribution range of Great Lakes lake sturgeon [3]. Despite substantial efforts to rehabilitate lake sturgeon, which include hatchery supplementation, their current abundance in the Great Lakes is estimated to be <1% of historical levels [1,3].
Despite substantial population declines, little is known about the infectious diseases of lake sturgeon compared to other sturgeon species (Order Acipenseriformes). This is a matter of concern given the propensity of disease to impede hatchery-based conservation and aquaculture productivity for sturgeon in North America [4,5] as well as worldwide [6,7]. In particular, viral diseases are important sources of mortality in a range of sturgeon species across North America, including white sturgeon (A. transmontanus), shortnose sturgeon (A. brevirostrum), pallid sturgeon (Scaphirhynchus albus), and shovelnose sturgeon (S. platorhynchus) [8][9][10][11][12].
Among the viruses associated with disease outbreaks in sturgeon, herpesviruses within the Family Alloherpesviridae have been identified as important pathogens of multiple sturgeon species [7,13,14]. The first report of a herpesvirus infection in sturgeon was from juvenile white sturgeon in California, USA, in 1990 [13]. This disease-causing virus, now known as Acipenserid herpesvirus-1 (AciHV-1; syn. white sturgeon herpesvirus 1), as well as Acipenserid herpesvirus-2 (AciHV-2; genus Ictalurivirus) [14], are both recognized as important sources of mortality in juvenile captive-reared white sturgeon in the western USA [13][14][15][16]. Subsequent studies have uncovered similar herpesviruses in Siberian sturgeon (A. baerri) in Russia [17,18], wild white sturgeon of the Columbia River (Oregon, USA) [19,20] and the Snake River (Idaho, USA) [19,20], and in cultured shortnose sturgeon from northeastern Canada [21]. Importantly, AciHVs have been isolated from the reproductive tissues of sexually mature and apparently healthy wild white sturgeon, raising the possibility of virus transmission from infected broodstock to offspring [14]. Despite this group of viruses having been detected in various North American sturgeon populations, an AciHV had never been detected in, or isolated from wild lake sturgeon. However, just prior to submission of the current manuscript, Walker et al. (2022) reported the detection of a novel alloherpesvirus in lake sturgeon in Wisconsin, USA, via a degenerate PCR assay and electron microscopy [22]. Although this virus, termed lake sturgeon herpesvirus, was found in association with skin lesions, in vitro isolation was not reported and its pathogenic potential was not determined.
Given the lack of reports of AciHV-1 and AciHV-2 in the Great Lakes basin in conjunction with their ability to cause sizeable mortality events in other sturgeon species, the Great Lakes Fishery Commission-Great Lakes Fish Health Committee designated these viruses (i.e., "white sturgeon herpesvirus") as Emergency Fish Pathogens in the 2014 Model Program for Fish Management in the Great Lakes [23]. However, during a study investigating the infectious diseases of Great Lakes lake sturgeon conducted herein, a replicating agent with acipenserid herpesvirus-like characteristics was initially detected and subsequently isolated from grossly appreciable skin lesions in adult lake sturgeon from the Lake Huron and Lake Erie watersheds of the Great Lakes basin. As such, the objectives of this study following disease surveillance efforts were to initially characterize and identify the replicating agent and investigate the tissue changes associated with naturally occurring infections in adult lake sturgeon. Following the successful isolation of this alloherpesvirus, an additional goal was to characterize its capacity to cause disease and/or mortality in juvenile lake sturgeon via in vivo challenge experiments under controlled laboratory conditions.

Fish and Sampling
During May and June of 2019 (year one), adult lake sturgeon were sampled from two locations within the Great Lakes basin: (a) spawning or near-spawning phase adult lake sturgeon, captured via hand netting by Michigan State University personnel in the upper Black River (Lake Huron watershed, Cheboygan County, MI, USA); and (b) adult non-spawning lake sturgeon, captured via trotline by Michigan Department of Natural Resources (MDNR) personnel in the St. Clair River (Lake Erie watershed, St. Clair and Macomb County, MI, USA).
Upon capture, fish were manually and gently restrained while partially immersed in water for gross examination (<3 min) and non-lethal tissue collections. Biometric data (i.e., length, weight, girth) were recorded, after which, whole blood was collected by caudal venipuncture. A portion of whole blood was immediately transferred into a sterile heparinized tube (Heparin sodium salt, ThermoFisher Scientific, Waltham, MA, USA) for subsequent packed cell volume (PCV) measurement and 1 mL of whole blood was placed into a 1.5 mL tube for virological analyses, both tubes were placed in an ice-chilled cooler. If fish were ready to spawn, reproductive fluids and/or eggs were manually expressed, collected from the cloaca via syringe, saved in a sterile 5 mL centrifuge tube or Whirl Pak™ (VWR, Radnor, PA, USA), and immediately placed on ice. When skin lesions of concern were observed, a small amount of tissue was biopsied (<250 mg) using forceps and either a sterile scalpel or scissors. The biopsied tissue was divided and saved in RNAlater (Sigma-Aldrich, St. Louis, MO, USA) on ice for molecular analyses, in a 2 oz Whirl Pak™ on ice for virus isolation, and in some cases, the biopsied tissue was saved in 10% buffered formalin for histopathological analyses. During the year one field season, all samples for virus isolation were frozen at −20 • C after collection and prior to further analyses.
Differences in prevalence of grossly observable virus-suspect skin lesion (see below) by fish sex collected from the Black River in 2019 and 2020 were evaluated using Fisher's Exact Test in Microsoft ® Excel ® (Version 2207 Build 16.0.15427.20182).

Hematology
Less than eight hours post-collection, heparinized blood from each fish was aliquoted into a micro-hematocrit tube. Whole blood was pelleted at 16,000 rpm in a CritSpin™ Hematocrit Centrifuge (Iris Sample Processing, Westwood, MA, USA) for 2 min and the packed cell volume (PCV) was measured following centrifugation [24].

Virus Screening and Isolation
Clarified supernatant was centrifuged just prior to inoculation (5000 RPM, 15 min, 4 • C) onto the cells. All samples from year one and two were inoculated onto EPC, CHSE, WSSK, and WSxLS, whereas a subset of samples from year one were inoculated onto WSGO. Use of the WSGO cell line was limited primarily due to cultivation challenges. After inoculation, all samples and cell lines were incubated at 15 • C. After a 14-day observation period, a second passage was performed on all samples, with no centrifugation step in between to allow for potential cell-to-cell virus transmission. Inoculated cell cultures were passed two to four more times for a total of 60 days on cells. If cytopathic effects (CPE) were noted, samples were passed into 25 cm 2 culture flasks containing fresh cells (<48 h old). If CPE persisted, supernatant and cells were preserved at −80 • C in a solution of 20% FBS and 20% glycerol and/or frozen un-diluted for further analyses.

Electron Microscopy
For transmission electron microscopy, representative skin lesions that had been fixed in neutral-buffered, 10% formalin solution were trimmed into 2 mm pieces and postfixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer for 2 h. Tissues were serially dehydrated in acetone and embedded in Poly/Bed 812 resin (Polysciences Inc., Warrington, PA, USA) in flat molds. Sections were obtained with a Power Tome XL ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA). To identify areas of interest, semithin (0.5-µm) sections were stained with epoxy tissue stain and examined under a light microscope. Ultrathin (70 nm) sections were then cut, mounted onto 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined under a 100 CXII transmission electron microscope (JEOL, Peabody, MA, USA). Similarly, WSxLS cells from cell culture samples (Section 3.4) exhibiting cytopathic effects (CPE) were harvested, centrifuged at 600 RPM (5 min, 15 • C) and the supernatant discarded. The cell pellet was then fixed in 10% buffered formalin and further processed as above.
2.6. Molecular and Gene Sequence Analyses 2.6.1. Nucleic Acid Extraction Representative skin lesion biopsies were processed for molecular analyses by thawing RNAlater (Sigma-Aldrich, St. Louis, MO, USA) preserved tissues at 4 • C, followed by centrifugation (5000 RPM, 10 min) to pellet the tissue and removal of stabilization buffer via micropipette. Following a single rinse with sterile phosphate-buffered saline (PBS), nucleic acid (DNA) extraction was performed using the DNeasy Blood and Tissue kit (Qiagen Inc., Germantown, MD, USA) according to the manufacturer's protocol for animal tissue. The concentration of extracted DNA was quantified via a Qubit™ fluorometer using the double-stranded DNA broad range assay kit (dsDNA BR Assay Kit; Invitrogen, Carlsbad, CA, USA). Likewise, cell culture samples showing CPE, along with noninoculated negative control cells, were harvested for DNA extraction and subsequent PCR analyses. In brief, once samples were passed and CPE observed (5-14 days post passage onto fresh cells), DNA was extracted from samples using the DNeasy Blood and Tissue kit following the manufacturers protocol for cultured cells.

Endpoint PCR Analyses
As the observed skin lesions were consistent with acipenserid herpesviral infections in other sturgeon species [13,14], degenerate PCR primers targeting a conserved region of the DNA polymerase gene of several alloherpesviruses were employed [34] using the cycling parameters of Kurobe et al. (2008) [18]. The HV (5 -CGG AAT TCT AGA YTT YGC NWS NYT NTA YCC-3 ) and Cons lower (5 -CCC GAA TTC AGA TCT CNG TRT CNC CRT A-3 ) primers amplify an~500 bp region of the DNA polymerase gene of several alloherpesviruses, including AciHV-1 and AciHV-2 [18]. Each 25 µL reaction was comprised of 12.5 µL 2 × GoTaq Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA), 5 µL of DNA template, and 40 pmol of each primer, with the remaining volume comprised of sterile, nuclease free water (Invitrogen, Carlsbad, CA, USA). Nuclease-free water, DNA extracted from apparently healthy and previously sampled adult lake sturgeon fin samples, as well as DNA extracted from uninoculated WSxLS cells served as template for negative control reactions, whereas NA extracts from previously confirmed AciHV-1 and AciHV-2 isolates were used as templates for positive control reactions. Each PCR was conducted in a Mastercycler Pro S 6325 Vapo protect PCR System (Eppendorf, Hamburg, Germany). PCR products were subsequently separated by gel electrophoresis in a 1.5% agarose gel infused with 0.0001% SYBR Safe DNA Gel Stain (ThermoFisher Scientific, Waltham, MA, USA; 1 mM) at 100 V for 30 min and then visualized via UV transillumination under a UVP DigiDoc-It Imaging System (UVP, Upland, CA, USA). A TrackIt™ 1 kb plus DNA ladder (Invitrogen, Carlsbad, CA, USA) was utilized for reference of amplicon size.

Gene Sequence Analysis
Representative cell culture and skin lesion samples yielding amplicons of~500 bp of the DNA polymerase gene target were purified using the QIAquick Spin Kit (Qiagen Inc., Germantown, MD, USA) and submitted to the Genomics Technology Support Facility (Michigan State University, East Lansing, MI, USA) for bidirectional Sanger sequencing using the HV and Cons primers of Hanson et al. (2006) [34]. Additionally, a subset of amplicons from PCR-positive cell culture and skin lesion samples were purified as above and subsequently cloned using the TOPO-TA cloning kit with TOP-10 Escherichia coli chemically competent cells (Qiagen Inc., Germantown, MD, USA) following the manufacturer protocols. Up to six resultant clones from each sample were screened for the target amplicon size via PCR analysis using primers M 13 Forward-20 (5 -GTA AAA CGA CGG CCA G-3 ) and M 13 Reverse (5 -CAG GAA ACA GCT ATG AC-3 ) and the following cycling parameters: 94 • C for 10 min, 25 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 45 s, and a final extension at 72 • C for 10 min. PCR products were visualized via gel electrophoresis as described above. Cloning-derived amplicons (n = 3-6 per sample) were purified, quantified, and submitted for sequencing as described above. Generated chromatograms were analyzed for quality and trimmed using Chromatogram Explorer (version 5.0.2.3). Contigs were assembled using the BioEdit sequence alignment editor (version 7.2.5) [35] via the Contig Assembly Program.

Nanopore Sequencing and Genomic Analyses
Genomic DNA was prepared using the Puregene DNA kit (Qiagen Inc., Germantown, MD, USA) following the manufacturer's protocol for cultured cells. Preparations from lake sturgeon herpesvirus isolate 200413-11TC, derived from a lesion sampled in an adult lake sturgeon in the Black River in year two (Section 3.4), were sequenced using a rapid barcoding kit (RBK-004, Oxford Nanopore Technologies, Oxford, UK) on a R9.4.1 flow cell. Raw Fastq sequences were filtered to a minimum read quality of 15 and a minimum length of 2kb using NanoFilt [36]. The filtered data, 220,725,293 bases in 57,034 reads, was assembled using Canu v2.0 [37]. The nanopore reads were re-mapped to the assemblies at a median 69-fold depth of coverage and consensus base calls were produced by Medaka v1.2.3 (https://github.com/nanoporetech/medaka [Accessed on 27 June 2022]) which produced two, non-overlapping contigs (119,069 and 87,627 bp).

Phylogenetic Analyses
Sanger generated sequences of the polymerase gene from discrete isolates were initially analyzed using the nucleotide Basic Local Alignment Search Tool (BLASTn) software from the National Center for Biotechnology Information (NCBI) nucleotide database. Reference sequences showing similarity were downloaded from NCBI, as were outgroups, and an alignment between study and reference sequences performed using ClustalW in MEGAX (Version 10.2.4) [40].
Similarly, MAFFT alignments were performed on nucleotide and translated amino acid sequences using the complete CDS of the 12 core alloherpesvirus ORFs from 17 alloherpesviruses for which complete or nearly complete sequences are available (Table S3) [22]. ML trees of the translated amino acid sequences for the complete DNA packaging terminase subunit 1 and DNA polymerase catalytic subunit CDS, as well as the 12 core alloherpesvirus genes were generated using the Le_Gascuel_2008 model with Gamma distribution and invariable rates (LG + G + I) with 100 replicates [41]. Phylogenetic analyses of core alloherpesvirus genes were performed in MEGAX and resultant trees edited and refined in FigTree v1.4.2, Adobe Illustrator and Adobe Photoshop. During May of 2020 and 2021, fish were spawned at the Black River Streamside Rearing Facility following previously described protocols and rearing practices [42][43][44][45][46]. In year one, fish were derived from four parental crosses, where parents were free of gross signs of disease. In year two, fish were derived from a single parental cross, where parents were free of gross signs of disease. Upon reaching eight weeks of age, fish were transported to the Michigan State University-University Research Containment Facility (MSU-URCF), where they were reared under quarantine conditions in a 680 L flow through tank supplied with ultraviolet-treated, oxygenated well water (11 • C ± 2 • C;~9 L/min). Fish were fed frozen blood worms (Brine Shrimp Direct, Ogden UT, USA) daily (~2-5% body weight), and the tank was cleaned and siphoned daily to remove detritus/uneaten food. Prior to virus exposure, a subset of fish (n = 60) were screened via tissue culture for the presence of viruses, including AciHVs, and verified to be free from infection via standard laboratory protocols [32]. Briefly, fish were euthanized in tricaine methanesulfonate (MS-222; Syndel, Ferndale, WA, USA; 250 mg/L) buffered with sodium bicarbonate (500 mg/L), tissues collected, processed, and inoculated onto cells as detailed in Section 2.5. Additionally, tissues from representative 5 fish pools were screened with the Hanson et al. PCR assay [34] with subsequent cloning of amplicons as described in Section 2.6.3. No virus was detected via PCR in representative pools of tissue prior to conducting virus-exposure experiments.

Preparation of Virus Inoculum for Exposure Experiments
Lake sturgeon herpesvirus isolate 200413-11TC was rapidly thawed from −80 •  and inoculating all dilutions onto 96 well plates (eight replicate wells per dilution), which were observed for the development of CPE for 21 days, at which point wells with CPE were counted and the TCID 50 calculated [47]. Virus was transferred on ice to MSU-URCF for exposure experiments.

Pilot Exposure Experiments
To guide a replicated virus exposure study, multiple small pilot experiments were undertaken. Juvenile lake sturgeon (n = five/treatment; mean weight 1.74 g, Table 1) were exposed to Great Lakes lake sturgeon herpesvirus isolate 200413-11TC via intracelomic (IC) injection or bath immersion and after exposure, were maintained in flow-through (water temperature of 11 • C + 2 • C;~1.5 L/min) or recirculating (water temperature of 22 • C ± 1 • C) 37.8 L aerated glass aquaria (Table 1). For IC injection exposure, fish were anesthetized with sodium bicarbonate-buffered (250 mg/L) MS-222 at a concentration of 125 mg/L and once sedate, injected with 25 or 50 µL (stock concentration: 1.53 × 10 2 -1.53 × 10 6 TCID 50 /mL) of virus infected or negative control cell suspensions (Table 1) using sterile 1 mL syringes and 25-gauge needles. Following injection, fish were released back into their experimental tanks and monitored for recovery. For pilot bath immersion exposures, fish were immersed in a 125 mL suspension of virus (treatment group; 6.40 × 10 5 TCID 50 /mL; Table 1) or cell growth media (negative control group) for 60 min at a concentration of one part virus suspension or negative control media to nine parts water (low dose) or one part virus suspension/media to four parts water (high dose). Following immersion exposure, fish and the virus or media suspensions were gently poured back into their respective 37.8 L flow-through glass aquaria.
Fish were monitored and cared for daily (Section 2.7.1). All dead fish were examined and multiple tissues (eye, fin, gill, kidney, spleen, heart, oral siphon, barbels, and/or skin lesions when present) aseptically collected, visceral organs and siphon/barbel samples pooled per fish, and processed as previously described for inoculation onto EPC, CHSE, WSSK, and WSxLS cell lines. Skin lesion samples were not pooled with other tissues and analyzed individually. Cell culture samples showing characteristic CPE observed on primary (i.e., first pass) inoculation were considered positive for viral infection. Terminally moribund fish and fish surviving until completion of the pilot experiments (i.e., 202-295 days post-challenge; Table 1) were euthanized via MS-222 overdose as above and analyzed similarly. All challenge experiments were conducted in accordance with the MSU-Institutional Animal Care and Use Committee (AUF: 201900057 and 202200033). Table 1. Summary data for pilot in vivo experiments in juvenile Great Lakes lake sturgeon (A. fulvescens; n = 5/treatment/pilot experiment) that were exposed to the newly isolated Lake Sturgeon herpesvirus under multiple conditions and exposure routes. TCID 50  Guided by results from pilot experiments, a larger scale and replicated immersion exposure experiment was undertaken. For experimental inoculum, virus was cultured and quantified as described above. Fish (mean weight of 1.20 g; n = 8 fish per replicate tank, n = 6 replicates) were immersed in a 120 mL suspension of lake sturgeon herpesvirus isolate 200413-11TC at a concentration of one-part virus suspension to three parts tank water, equating to an exposure dose of 1.28 × 10 6 TCID 50 /mL). Another six replicate tanks (n = 8 per replicate) were similarly mock challenged with one-part WSxLS cell/media suspension to three parts clean tank water with aeration. Following a 60 min immersion exposure, fish and the respective suspension were gently poured into their respective 37.8 L flow-through glass aquaria maintained at 11 • C ± 2 • C (flow rate of 1.5 L/min) with supplemental aeration. As detailed previously, dead fish were collected daily. Terminally moribund fish and fish surviving until the completion of the immersion challenge (112 days) were euthanized as described above and tissues (internal organs, siphon, barbel, fin, gill, lesions) collected for virus isolation and molecular analyses. Tissues for each fish were pooled as described above (visceral organs and siphon/barbel), however tissues from different fish were not pooled together. In brief, select cell culture supernatant samples showing CPE were analyzed using the primer set developed by Hanson et al. (2006) as described above [34], and a subset of samples were cloned and sequenced using methods described in Sections 2.5 and 2.6.1-2.6.3.

Gross Findings in Adult Great Lakes Lake Sturgeon
During year one, 137 spawning or near-spawning adult lake sturgeon (n = 111 males, n = 26 females) of varying lengths and weights ( Table 2) (Table 2). Likewise, among the 76 adult lake sturgeon examined from the St. Clair River in year one, one fish (1.3%; length of 120.8 cm, weight of 41.4 kg, and girth of 49.2 cm) had grossly similar skin lesions on the body and head ( Figure 1d and Table 2). During year two, 79 adult spawning or near-spawning lake sturgeon (n= 54 males, n= 25 females) of varying lengths and weights (Table 2)  When comparing the presence of skin lesions in males and females in 2019 using Fisher's Exact Test, a one-tailed p-value of 2.42 × 10 −23 was observed, indicating a significant difference in lesion prevalence. However, in 2020, a one-tailed p-value of 0.1371 was observed, indicating lesions prevalence was not significantly different by sex.

Hematology
Fish from all sites in year one and two had a mean PCV of 32.2%, with some variation by sampling site and year (Table 2). Overall, fish presenting with the skin lesions had a mean PCV of 33.9%, compared to a mean PCV of 32.6% in fish without skin lesions ( Table 2). No clear trends in PCV were appreciable by site or year (Table 2); however, female lake sturgeon had lower mean PCVs in both year one and two at the Black River site when compared to males (

Histopathology
Sections of fin, gills and the site of gross lesions were examined from five fish sampled from the Black River in year two. Fins and gills (lesions not collected for histopathology) were examined from two additional fish. All seven had skin abnormalities suspect for Acipenserid herpesviruses and were from the same site and year (Black River, year two). None of the fish had lesions in the gills. The microscopic changes in the gross lesion site were most severe in one fish of the five where lesion tissue was available, but overall similar in the other four fish. There was marked ballooning degeneration of the deeper portion of the stratum spinosum of the epidermis (Figure 2a). Affected cells were enlarged with lightly eosinophilic to clear cytoplasm and their nuclei were swollen with marginated chromatin (Figure 2b). Within the nuclei, there was often lightly eosinophilic, flocculent material forming central inclusions that displaced the chromatin. The more superficial portion of the epidermis had undergone necrosis and there was loss of nuclear detail, marked karyorrhexis and sloughing of epithelial cells. Multifocally there was transmural necrosis also affecting the superficial portion of the underlying lepidotrichia (Figure 2c). In the subepidermal portion of the connective tissue surrounding the lepidotrichia was loss of cellular detail and multiple foci of karyorrhexis. In the deeper portions of the connective tissue separating the mineralized spines was extensive neovascularization and proliferation of fibroblasts (granulation tissue) with dense infiltrates of small lymphocytes (Figure 2d). Multiple segments of the mineralized spines were partially lysed, had scalloped edges and were surrounded by proliferating fibroblasts or were completely lost in other areas (Figure 2e). There was common clustering of pigmented cells in the superficial connective tissue. In all submitted skin samples, there was marked epidermal hyperplasia with severe intercellular edema (spongiosis) in the stratum spinosum. Keratinocytes focally lost orientation, forming small clusters of cells, and mitotic figures were common. There were dense infiltrates of small lymphocytes in the lower portion of the stratum spinosum focally extending into the stratum basale (Figure 2f). Lymphocytes were commonly surrounded by a clear halo and surrounded individual necrotic keratinocytes. There was often clustering of pigmented cells within the superficial stratum spinosum of the more normal appearing epidermis forming a transition zone to the deeper portions of the epidermis that were infiltrated by lymphocytes.

Cell Culture
In year one, 119 whole blood, 91 reproductive fluid, and 8 skin lesion samples collected from the Black River and 57 whole blood, 3 reproductive fluid, and 1 skin lesion samples collected from the St. Clair River were inoculated onto the EPC, CHSE, WSSK-1, and WSxLS cell lines, with a subset also being inoculated onto WSGO. After a 60-day observation period, there was no observable CPE in any of the samples on any cell lines. In year two, 67 whole blood, 47 reproductive fluid, and 12 skin lesion samples collected from the Black River were inoculated onto EPC, CHSE, WSSK-1, and WSxLS. No CPE was detected after 60 days in any whole blood or reproductive fluid samples, nor in any of the skin lesion samples inoculated onto the WSSK, EPC, or CHSE cell lines. In contrast, CPE, in the form of large, multinucleated cells (i.e., syncytia), were observed on the WSxLS cell line (Figure 3) in 11 of the 12 skin lesions (91.6%), beginning as early as five days post inoculation. By two weeks post-inoculation, all cells had either detached, lysed, or formed syncytia. Subsequent passages resulted in identical syncytia formation and individual isolates were cultured and cryopreserved for further analyses.

Electron Microscopy
Transmission electron microscopy (TEM) revealed large numbers of herpesvirus-like particles (~85 nm) with electron-dense nuclei within the degenerating nuclei of affected cells (Figure 4a

Electron Microscopy
Transmission electron microscopy (TEM) revealed large numbers of herpesvirus-like particles (~85 nm) with electron-dense nuclei within the degenerating nuclei of affected cells (Figure 4a (Table 3) yielded sequences ranging from 468-529 bp and comparisons of quality trimmed (i.e., 458-477 bp) sequences using BLAST revealed highest similarity (ranging from 96.2-100%) to the newly detected lake sturgeon herpesvirus Wolf River strain (GenBank Accession Number: OK485036), followed by 79.0-82.0% similarity to an AciHV-1 DNA polymerase gene reference sequence (GenBank Accession Number: EF685904.1; Table 3). When representative amplicons from both sampling years and sites (3 from year one and 3 from year two) were cloned and subsequently sequenced (3-6 clones per sample), the generated sequences all were most similar to the same newly detected lake sturgeon herpesvirus Wolf River strain (GenBank Accession Number: OK485036; 98.7-100%; Table 3), followed by 81.0-82.0% similarity to an AciHV-1 DNA polymerase gene reference sequence (GenBank Accession Number: EF685904.1).

In Vivo Challenge Experiments
Following small-scale pilot challenges of juvenile lake sturgeon to multiple concentrations of the newly isolated lake sturgeon herpesvirus via multiple exposure routes (Table 1), gross disease signs, in the form of decreased reactivity to feed, lethargy, and peri-oral hemorrhage (Figure 6a), began to manifest at 3-31 days post-exposure (PE). Other signs of disease observed throughout the pilot studies included enophthalmia and focal dermal ulceration on the head and/or body of the fish (Figure 6b,c). Eventually, severe lethargy that progressed to fish lying in dorsal recumbency, severe ulceration of the caudal fin into the peduncle with associated hemorrhage (Figure 6d), and/or mortality began on day 8 PE and continued until day 84 PE (Table 1). Tissues from representative, grossly apparent skin lesions in both immersion and IC injection pilot experiments were inoculated onto WSxLS cells. After 7 days, syncytia were observed consistent with CPE from cells inoculated with lesion tissues collected from adult lake sturgeon. There was no evidence of CPE on CHSE, EPC, or WSSK, nor was virus recovered from other tissues sampled (i.e., oral siphon, barbel, eye, visceral organs). dal fin into the peduncle with associated hemorrhage (Figure 6d), and/or mortality began on day 8 PE and continued until day 84 PE (Table 1). Tissues from representative, grossly apparent skin lesions in both immersion and IC injection pilot experiments were inoculated onto WSxLS cells. After 7 days, syncytia were observed consistent with CPE from cells inoculated with lesion tissues collected from adult lake sturgeon. There was no evidence of CPE on CHSE, EPC, or WSSK, nor was virus recovered from other tissues sampled (i.e., oral siphon, barbel, eye, visceral organs). Figure 6. Gross disease signs observed in juvenile lake sturgeon (Acipenser fulvescens) exposed to the newly isolated lake sturgeon herpesvirus via intracolemic injection (a,c) or immersion bath (b,d); isolate 200413-11TC). (a) Hemorrhage surrounding the oral siphon extending linearly to the barbels Based in part on results from pilot experiments and experimental exposure studies with other acipenserid herpesviruses [13,14], an immersion route of exposure was selected for a larger scale, replicated challenge experiment. In the replicated immersion challenge, fish were exposed to lake sturgeon herpesvirus isolate 200413-11TC at a concentration of 1.28 × 10 6 TCID 50 /mL, after which gross signs of disease were noted in virus-exposed fish beginning 10 days PE. Early signs of disease included moderate to severe lethargy when compared to negative controls across all replicates, and overall decreased reactivity to feed. Additionally, hemorrhaging around the oral siphon began 10 days PE (Figure 7a). At 15-16 days PE, more gross signs of disease began to manifest, including mild corneal opacity and mild-moderate erosion of the caudal fin. Within 48 h of caudal fin erosion onset, a subset of affected fish had near complete loss of the caudal fin and ulceration extending deep into the caudal peduncle musculature, often accompanied with moderate-severe hemorrhaging (Figure 7b,c). Disease signs continued to progress to marked emaciation, more widespread hemorrhaging around the oral siphon and caudal peduncle, and complete loss of the caudal fin and posterior portions of the caudal peduncle (Figure 7d). No gross signs of disease were noted in negative control, mock-exposed fish. Mortality in virusexposed fish began 19 days PE and continued through 76 days PE, where all dead or terminally moribund and euthanized fish had grossly similar ulcerations of the caudal peduncle and resultant near complete loss of the caudal fin. Following conclusion of this experiment at 112 days PE, cumulative percent mortality (CPM) across all virus-exposed replicates reached a mean of 33.3% (Figure 8) compared to a mean CPM of 0.0% across all negative control, mock-exposed replicates.
No gross signs of disease were noted in negative control, mock-exposed fish. Mortality in virus-exposed fish began 19 days PE and continued through 76 days PE, where all dead or terminally moribund and euthanized fish had grossly similar ulcerations of the caudal peduncle and resultant near complete loss of the caudal fin. Following conclusion of this experiment at 112 days PE, cumulative percent mortality (CPM) across all virus-exposed replicates reached a mean of 33.3% (Figure 8) compared to a mean CPM of 0.0% across all negative control, mock-exposed replicates.   Upon inoculation of collected tissues onto the WSxLS cell line, CPE (i.e., formation of syncytia) identical to what was observed in the herpesvirus infected adult lake sturgeon developed in representative skin lesion samples. Cell cultures inoculated with other tissues (i.e., siphon, barbel, visceral organs) produced no CPE. Subsequent PCR analyses of representative cells showing CPE using the degenerate primer set described above [34] yielded an ~500 bp amplicon; following cloning and Sanger sequencing, recovered virus isolates were confirmed to be the same lake sturgeon herpesvirus to which the fish were (i.e., siphon, barbel, visceral organs) produced no CPE. Subsequent PCR analyses of representative cells showing CPE using the degenerate primer set described above [34] yielded an~500 bp amplicon; following cloning and Sanger sequencing, recovered virus isolates were confirmed to be the same lake sturgeon herpesvirus to which the fish were exposed, partially fulfilling Rivers' postulates. There was no evidence of an active replicating agent in cells inoculated with tissues collected from survivors at the end of this study. Tissues from survivors were not analyzed by PCR.

Discussion
Herein, we describe the first in vitro isolation and virulence assessment of an alloherpesvirus from wild lake sturgeon in general, as well as the first detections of an alloherpesvirus in lake sturgeon inhabiting the Lake Huron and Lake Erie watersheds of the Great Lakes basin, North America. These findings are significant for several reasons. First, Great Lakes lake sturgeon populations are estimated to be <1% of historical abundances, and substantial resources are currently being allocated to the restoration of this iconic Great Lakes fish [1]. In this context, the present study not only demonstrated the presence of actively infectious virus was associated with skin lesions in wild adult lake sturgeon, but also that the newly isolated alloherpesvirus can induce severe disease and subsequent mortality in experimentally exposed juvenile lake sturgeon. These findings have both economic and environmental consequences, as these wild sturgeon serve as gamete sources for hatchery-based conservation and restoration efforts for these endangered animals in the Great Lakes region. Although this virus was not recovered from reproductive fluids in the current study, other alloherpesviruses from sturgeons (i.e., AciHV-1 and -2, white sturgeon) have been isolated from reproductive fluids of spawning adults with gross signs of disease [14], raising suspicions of possible transmission to and subsequent negative effects in offspring. Whether this newly detected virus has been or is leading to disease and/or mortality in wild and hatchery reared juvenile lake sturgeon remains unknown and warrants priority attention.
Second, other acipenserid herpesviruses (formerly known as white sturgeon herpesviruses) [13,14] are currently listed as emergency pathogens in the Great Lakes Fishery Commission-Great Lakes Fish Health Committee Fish Health Model Program [23]. This list, curated based on the potential threats certain pathogens pose to Great Lakes fishes, contains several significant fish pathogens believed to not yet be in the Great Lakes basin. As such, isolation of this virus is highly relevant to Great Lakes lake sturgeon management and may necessitate revision of the current list of emergency pathogens.
Third, alloherpesviruses have immunomodulatory effects in other fishes, leading to immunosuppression and increased potential for opportunistic infections [63,64]. For example, previous work found when common carp (Cyprinus carpio) were experimentally infected with Cyprinid herpesvirus 3, infection negatively affected the mucosal barrier of the skin and associated immune system activity [63]. Furthermore, infection with Salmonid herpesvirus 3 (EEDV) in lake trout (Salvelinus namaycush) induced skin ulcerations which were co-colonized with multiple fish-pathogenic bacteria, including Flavobacterium psychrophilum and Aeromonas spp. [64]. Similarly, Acipenserid herpesvirus 2 has also been linked to co-infections with Streptococcus iniae in white sturgeon (A. transmontanus) [16]. Collectively, literature supports the ability of alloherpesviruses to both negatively affect the immune system and subsequently provide opportunity for secondary or co-infections with other fish pathogens [16,63,64].
Lastly, herpesviruses are known to establish latent and chronic infections in hosts, where stressors, such as spawning or environmental factors could lead to recrudescent infections [64,65]. This was demonstrated during the resurgence of salmonid herpesvirus 3 (EEDV; Alloherpesviridae) in hatchery-reared lake trout (S. namaycush), where disease and mortality attributed to EEDV was thought to be induced by a combination of environmental stressors (e.g., temperature fluctuations and heavy rainfall) in addition to rearing densities [64]. Whether similar stressors and immunomodulatory effects are associated with infection emergence, status, or severity in the context of this newly isolated alloherpesvirus remains to be determined. Similarly, whether or not this alloherpesvirus establishes latency as other herpesviruses do is presently unknown [64]. Further, it is unclear if the targeted tissues and diagnostic tests deployed herein would have detected any latent infections. Consequently, investigating tissue tropism of the virus and documenting when and if it establishes a latent state would help optimize latent infection diagnostics [64].
Although the present study reports the first isolation of an alloherpesvirus from Great Lakes lake sturgeon, Walker et al. (2022) recently reported the molecular detection of an alloherpesvirus in four rivers in Wisconsin (US), including within the Lake Michigan watershed [22]. Following initial PCR screening for the alloherpesvirus DNA polymerase gene [34] and subsequent genome sequencing and assembly, Walker et al. (2022) determined the virus reported in WI lake sturgeon was an alloherpesvirus most similar to, yet distinct from AciHV-1. They proposed the name lake sturgeon herpesvirus to represent this newly characterized virus [22,34]. Interestingly, using the same degenerate primer set [34] and subsequent sequence analysis of partial DNA polymerase gene sequences, the virus detected in the present study was similar (~93% homology), yet not identical, to the lake sturgeon herpesvirus Wolf River strain [22,34]. Additional comparison of the genomes generated by Walker et al. (2022) and this study revealed high similarity across a small (i.e., 156 bp) portion of the DNA packaging terminase subunit I, yet shared <90% similarity across the entire CDS. Similarly, when compared across the other 10 core alloherpesvirus genes, the level of homology averaged 81.8% (range 68.9-86.3%). This was consistent with ANI comparisons across the available genomic data for the two lake sturgeon viruses, which revealed only 84% similarity using multiple alignment strategies (Table 5). Considering these results and acknowledging that strict criteria to delineate alloherpesvirus taxa are not fully established, we posit the genomically characterized virus detected in the current study is most similar to, yet distinct from, the lake sturgeon herpesvirus recently described by Walker et al. (2022) [22]. In fact, the %ANI between the lake sturgeon herpesvirus Wolf River strain detected by Walker et al. (2022) and the current study is~10% lower than the %ANI for two catfish alloherpesviruses (i.e., channel catfish virus and blue catfish alloherpesvirus; Table S4) that are believed to represent distinct taxa. Indeed, work done by Venugopalan et al. further supported the separation of channel catfish virus and blue catfish alloherpesvirus into separate taxa through host-specificity and serum neutralization studies [66]. Therefore, we propose the virus described in Walker et al. (2022) and the virus described herein be, respectively referred to as lake sturgeon herpesviruses 1 and 2 pending additional taxonomic investigations [22].
It is important to note that the geographic locales where these two viruses were recovered contain two genetically differentiated groups of lake sturgeon in the Great Lakes [67,68]. Furthermore, there is little mixing of individuals in Lake Michigan (Michigan vs. Wisconsin waters) [69], raising intriguing questions regarding the potential divergence of the two viruses since the potential lake sturgeon colonization of Lake Michigan from glacial refugia approximately 12,000 years ago [70].
Lake sturgeon herpesvirus 2 was detected in infected adult lake sturgeon residing in two (i.e., Huron and Erie) Great Lakes watersheds, where the virus was associated with grossly apparent skin lesions. Of note, these lesions were not only grossly and microscopically similar to those reported by Walker et al. (2022) [22], but also to white sturgeon infected with AciHV-2 [13]. Thus, it appears these viruses may have a similar disease manifestation despite representing different viral species and infecting different hosts. In the present study, the prevalence of these herpesvirus-linked skin lesions varied by sex in the Black River during both study years, whereby lesions were 2.2-3.4 times more prevalent in females than males. Infections with other fish pathogens (e.g., A. salmonicida and Carnobacterium maltaromaticum) in other fish species nearing and/or undergoing spawning have also been documented as more prevalent in females [71,72]. Potential factors behind the observed differences in virus-linked lesion prevalence by sex could involve the complex relationship between stress, sex, and the immune system [73]. Not only have female fish been documented to have higher cortisol levels during spawning that may dampen the immune response, but evidence also suggests female spawning fish have a less robust immune response compared to males [71][72][73][74]. Additionally, differences in the herpesvirus-linked skin lesion prevalence were apparent by sampling site, where prevalence was lower in the St. Clair River than the Black River in 2019 (1.1% vs. 6.6%; Table 2). Whether this relates to differences in spawning behavior, environmental factors and/or differences between the two sturgeon populations is unclear. It is noteworthy that in the Black River, nearly all fish were at the spawning phase, whereas fish in the St. Clair River were primarily pre-spawning phase, and spawning stress has been linked to emergence of active herpesvirus infections from latency and decreased immune competence [64,75,76].
Hematologic analyses revealed no significant differences in PCVs between fish with LSHV-associated lesions and those without. There is little published information regarding the relationships between hematologic parameters, such as packed cell volume, and alloherpesvirus infection. Overall, this study found no substantial differences in PCV between male and female fish from any site in any year, and all PCV averages fell within published sturgeon (A. fulvescens and A. gueldenstaiedtii) reference intervals (17-38% and 16-34%) [48,49].
To clarify the effects the newly recovered lake sturgeon herpesvirus may have on health and survival of juvenile lake sturgeon, in vivo laboratory challenge experiments were completed. Results indicate the newly isolated LSHV-2 is indeed capable of causing disease and subsequent mortality in juvenile lake sturgeon immersed in a virus-laden suspension under laboratory conditions. Likewise, these experiments revealed LSHV-2 was capable of replicating in naïve hosts and could be re-isolated from the resultant skin lesions, collectively fulfilling Rivers' postulates [77]. Notably, the progression of grossly apparent disease signs was remarkably consistent across virus-exposed replicates, whereby fish developing gross disease signs showed lethargy and decreased reactivity to feed, which eventually progressed to complete loss of the caudal fin and deep ulceration into the caudal peduncle in every fish that died (n = 16). Somewhat surprisingly, the gross disease signs in LSHV-2-exposed fish showed uncanny similarity (e.g., ulcerative lesions) to those often associated with Flavobacterium columnare, the causative agent of columnaris disease [78,79]. However, tissues from caudal ulcerations were collected from representative fish in all replicate tanks and inoculated onto Hsu-Shotts medium using routinely employed methods [32,80] and no yellow pigmented bacteria, including F. columnare, were recovered (data not shown). Considering these findings, fishery managers and fish health professionals/clinicians should be aware that LSHV-2 appears to induce disease signs that could be readily confused with columnaris disease, potentially leading to delayed/misdiagnosis and/or ineffective treatments, especially without deployment of species-specific in vitro or specialized molecular techniques.
This study is the first to successfully isolate and propagate in vitro, a lake sturgeon herpesvirus. The CPE induced by the LSHV (i.e., syncytia formation) was highly similar to that noted in AciHV 1 and 2 [13,14,19]. Notably, propagation of this alloherpesvirus was only possible using a cell-line that included cells derived from the original host, the lake sturgeon, and is consistent with reports of strong host specificity often exhibited by alloherpesviruses [81]. Based on the present study's results, it is tempting to speculate that LSHV-2 exhibits relatively narrow tissue tropism (e.g., the skin) given that it could only be recovered from the skin lesions of experimentally exposed fish; however, the sensitivity/limit of detection of the hybrid sturgeon cell-line that was utilized in this study is unknown and it is possible that a more sensitive assay might uncover virus presence in other tissues. Nevertheless, skin lesions clearly represent a priority tissue target for detecting/isolating LSHVs. In addition to requiring specialized cells and reagents, results from the current study affirm protocols established by the World Organisation for Animal Health (OIE), in that attempts to isolate an alloherpesvirus in vitro should avoid freezing of tissues [82]. Additionally, the use of a viral transport medium formulated based on recommendations from the American Type Culture Collection and American Fisheries Society-Fish Health Section [32,83] also seems to have enhanced successful virus recovery and may relate to the importance of antibiotics and fetal bovine serum for virus stability and replication. Based upon the results herein, when attempting to recover LSHVs in vitro, we recommend the following process be followed: (a) collect tissue and fluid samples directly into viral growth media (<1:4; W:V) detailed in Section 2.1; (b) maintain virus at 4 • C (i.e., unfrozen) and inoculate onto cells within five days of collection (less than 48 h is preferred); and (c) utilize cell lines derived, at least in part, from lake sturgeon.

Conclusions
This study describes the first isolation of an alloherpesvirus from wild adult lake sturgeon that concurrently exhibited gross cutaneous skin lesions. The recovered virus proved virulent to naïve juvenile Great Lakes lake sturgeon, whereby immersion exposure resulted in the development of disease and subsequent mortality and re-isolation of the virus from the skin lesions of affected hosts. Genetic and genomic characterization of the virus detected in this study revealed it was most similar but distinct from a very recently described LSHV (Walker at al. 2022) [22]. Based upon these results and until further taxonomic studies are undertaken, we propose the virus detected in Walker et al. and the virus isolated herein be referred to as lake sturgeon herpesvirus 1 and 2, respectively. Although much remains unknown about the effects LSHV-1 and 2 may have had or be having on LST populations, reports of the closely related Acipenserid herpesviruses being detected in reproductive fluids raises the potential for transgenerational transmission in lake sturgeon with unknown consequences for wild populations and hatchery conservation efforts alike. Research conducted in the present study is paving the way for ongoing and future research efforts seeking to arm those rearing lake sturgeon in the Great Lakes with efficacious management tools to reduce the negative health risks this virus poses to Great Lakes lake sturgeon populations and to their restoration.