Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

Historically, EHDV serotypes 1 and 2 have been detected throughout wild WTD populations of North America [21,23]. In 2006, an EHDV serotype 6 strain was isolated for the first time from moribund and dead WTD in Indiana and Illinois [22]. From 2006 to 2015, EHDV serotype 6 was isolated from WTD in 16 additional states (including Florida) and is now considered endemic throughout the central and eastern U.S. [4,15,22]. In recent years, EHDV-6 infections have surpassed EHDV-1 in WTD in the country [15]. Comparative genomic analyses of these U.S. EHDV-6 strains (hereafter referred to as U.S. endemic reassortant EHDV-6 strain (Indiana)) revealed that they represent reassortants, with segments 2 and 6 (encoding the serotype determining VP2 and VP5) derived from an exotic EHDV-6 strain (CSIRO 753) and the remaining eight segments derived from a U.S. endemic EHDV-2 strain [22,61]. The exotic EHDV-6 strain (CSIRO 753) was isolated from sentinel cattle in Australia (hereafter referred to as the Australian prototype EHDV-6 strain (CSIRO 753)) [62]. Later studies revealed that another reassortant EHDV-6 strain was circulating in cattle from Trinidad (hereafter referred to as the Trinidad reassortant EHDV-6 strain) [16,63]. The Trinidad reassortant EHDV-6 strain was argued to be the product of assortment events involving the Australian prototype EHDV-6 strain (8 segments), an Australian EHDV serotype 2 strain (segment 4 encoding the VP4 gene), and an EHDV-1 strain of unknown origin (segment 8 encoding the NS2 gene) [63]. The Trinidad reassortant EHDV-6 strain may also be circulating in cattle from neighboring Caribbean islands (Guadeloupe and Martinique) and the South American mainland (French Guiana, Ecuador) [16,[63][64][65]. Although the details of how and when the EHDV-6 serotype arrived in the Western Hemisphere remain obscure, it has been suggested that an Australian prototype EHDV-6 strain may have first arrived in the Caribbean and then spread to the U.S. where it donated segments 2 and 6 (encoding VP2 and VP5) in the creation of the U.S. endemic reassortant EHDV-6 strain now circulating in WTD populations in the central and eastern U.S. [15,22,61]. To date, neither the Australian prototype EHDV-6 strain nor the closely related Trinidad reassortant EHDV-6 strain have been detected in the U.S. in either WTD or cattle populations [15,22,61].
In this study, we report the isolation and genomic characterization of the same novel reassortant EHDV-6 strain from dead WTD on a Florida farm in 2019. The observed gross and microscopic pathology and other ancillary diagnostic results supported EHDV-6 as the likely cause of the observed disease in the farmed WTD. A manual RNAscope ® in situ hybridization assay targeting the VP1 gene demonstrated labeling of EHDV-6 nucleic acid in the heart, kidney, liver, and lung tissues. Genetic and phylogenetic analyses were conducted to compare this U.S. novel reassortant EHDV-6 strain from WTD to other EHDV strains including EHDV-6 strains isolated from WTD (e.g., U.S. endemic reassortant EHDV-6 strain) and cattle (e.g., Trinidad reassortant EHDV-6 strain and Australian prototype EHDV-6 strain). This study was conducted as part of the University of Florida (UF) Cervidae Health Research Initiative (CHeRI) program, aiming to characterize pathogens negatively impacting farmed WTD in Florida.

Outbreak, Clinical History, and Sample Collection
From 11 September 2019 to 9 November 2019, 12 WTD died or were euthanized on a farm located in Lake County, FL, U.S. The carcasses were necropsied, and each animal was individually identified with a unique number and the prefix "OV", which denotes O. virginianus. The clinical information for each animal is provided in Table 1.
Necropsies were performed by deer farmers or UF technicians following guidelines provided by the CHeRI (https://wec.ifas.ufl.edu/cheri/diagnostics/ (accessed on 29 March 2022)). During the field necropsies, samples of the heart, kidney, liver, lung, spleen, and whole blood were collected and transported on ice prior to fixing in 10% neutralbuffered formalin for histologic processing and/or freezing at −80 • C for diagnostic virology. Lung tissues from animals OV1224, OV1248, OV1289, OV1296, OV1300, OV1314, OV1317, and OV1324 were collected and submitted to the UF Microbiology, Parasitology, and Serology Diagnostic Laboratory of the College of Veterinary Medicine for bacterial and mycological isolation and identification. Samples of the heart, kidney, liver, lung, small intestine (only available for animal OV1248), and spleen from animals OV1248 and OV1296 were fixed in 10% neutral-buffered formalin and submitted for histologic processing and histopathologic examination at the UF Veterinary Diagnostic Laboratories. Following routine processing, 3-µm sections of the formalin-fixed, paraffin-embedded tissue samples (listed above) were stained with hematoxylin and eosin (H&E). This work was approved by the UF Institutional Animal Care and Use Committee (IACUC Protocol Numbers 201609390 and 201909390).

RT-PCR Detection of BTV, EEEV, EHDV, and WNV vRNA
Prior to extracting virus RNA (vRNA) from the heart, kidney, liver, lungs, and spleen samples, the tissues (previously stored at −80 • C) were thawed, aseptically minced using forceps, then homogenized to generate 10% w/v cell-free suspension homogenates in sterile phosphate-buffered saline (PBS) using a sterile manual tissue grinder (Fisher Scientific, Waltham, MA, USA). The resulting homogenates were subsequently cleared of debris by low-speed centrifugation (5 min at 1500× g) and aseptically transferred to sterile polypropylene centrifuge tubes. vRNA was extracted from virions in the tissue homogenates and whole blood samples using a QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. RNA extracts from blood and tissue homogenates of all animals were then screened for BTV and EHDV using a multiplex quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) assay as previously described [66]. For EHDV-positive homogenates, EHDV serotype was determined using a multiplex RT-PCR assay as previously described [67]. vRNA extracted from spleen homogenates were also tested for eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) using a VetMAX Plus One-Step RT-qPCR kit (Applied Biosystem), as previously described [68,69]. Briefly, 25-µL reactions containing 12.5 µL 2X RT-PCR Buffer, 1 µL 25X RT-PCR enzyme, RNAase-free water, 1 µL Xeno VIC assay primers and probe, and 0.1 µL Xeno RNA (internal positive control), to which was added 25 pmol (for WNV) or 19 pmol (for EEEV) of each primer and 5 pmol of probe: fluorophore 6-FAM, quencher BHQ1, resulting in a 21 µL master-mix. Thereafter, 4 µL of vRNA was added to the master mix containing either WNV or EEEV specific primers and probes, and RT-PCRs were performed in an Applied Biosystems 7500 fast Real-Time PCR System as follows: reverse transcription step at 48 • C for 10 min, initial denaturation step at 95 • C for 10 min followed by 40 cycles of 2-step cycling consisting of 95 • C for 15 s, and annealing/extension at 60 • C for 45 s.

RNAScope ® In Situ Hybridization (ISH) Assay
A manual in situ hybridization (ISH) assay using RNAscope ® technology was used to detect EHDV-6 RNA in formalin-fixed paraffin-embedded (FFPE) heart, kidney, liver, and lung tissues from animals OV1248 and OV1296. Specific probes were designed by Advanced Cell Diagnostics (Newark, CA, USA), based on the following genes: dihydrodipicolinate reductase gene (dapB; negative control probe) [ The ISH assay was performed using an RNAscope ® 2.5 HD Red Detection Kit (ACD, Hayward, CA) following the protocol provided by the manufacturer [70]. Three sections of 4 µm were cut from each paraffin block and mounted on Fisherbrand SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA). The slides were subjected to deparaffinization, target retrieval, protease digestion, and endogenous enzyme block. Probe hybridization was performed, and each of the three sections of a block was treated with a specific probe: one section received the dapB probe, a second section received the OV cytB probe, and a third section received the EHDV-6 VP1 probe. After probe hybridization, signal amplification, fast red chromogenic development, and counterstaining with hematoxylin was performed. Incubations were conducted in a humidity chamber and a HybEZTM oven (ACD, Hayward, CA, USA). After the slides were dried and mounted, they were examined for microscopic lesions using an Olympus BX53 light microscope (Olympus, Center Valley, PA, USA).

Virus Isolation in Cultured Cells
Virus isolation was attempted using the Aedes albopictus (Asian tiger mosquito) cell line C6/36 (ATCC CRL1660) for all animals except OV1299 as previously described [4]. Spleen samples were thawed and homogenized in sterile phosphate-buffered saline (PBS), using a sterile manual tissue grinder (Fisher Scientific) to generate 10% w/v cell-free homogenates. The suspension was cleared of debris by low-speed centrifugation (5 min at 1500× g). The supernatant was then filtered through a sterile 0.45 µm pore-size polyvinylidene fluoride filter (Fisher Scientific, Cat. Number 09-720-4). A 0.5 mL aliquot of the filtrate was inoculated onto confluent C6/36 cells in 25 cm 2 culture flasks. The cells were re-fed every 3 days and observed daily for virus-induced cytopathic effects (CPE) for 30 days. Non-inoculated cells were maintained in parallel as negative controls. When no CPE was observed by day 30 post-inoculation, a second passage was performed, and the cells were observed for another 30 days before being considered negative for virus isolation. After CPE were observed in 50% of the infected cells, they were scraped and harvested along with the spent cell growth medium and stored at −80 • C for further analyses.

Next-Generation Sequencing (NGS)
The frozen spent cell-growth media from samples were thawed and spun to remove cellular debris prior to extracting vRNA from virions using a QIAamp Viral RNA Mini kit (Qiagen) according to the manufacturer's protocol. cDNA libraries were generated using a NEBNext Ultra RNA Library Prep kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq sequencer (Illumina). Host (Aedes albopictus) sequences (GenBank accession number MNAF00000000.2) were removed using Kraken v2.0 (Johns Hopkins University School of Medicine, Baltimore, MD, USA) [71]. After removing the host sequences, the de novo assembly of the remaining paired-end reads was performed using SPAdes 3.5.0 [72]. The assembled contigs were then subjected to BLASTX searches against the National Center for Biotechnology Information (NCBI) nonredundant protein database, using OmicsBox v1.2.

Phylogenetic and Genetic Analysis
Maximum Likelihood (ML) phylogenetic trees were constructed based on separate nucleotide alignments of each of the 10 coding sequences of the EHDV isolated from OV1321 to 66 other EHDVs and one BTV (outgroup), whose sequences were available in the NCBI GenBank database (Supplemental Table S1). The alignments were performed using the MAFFT server (https://mafft.cbrc.jp/alignment/server/ (accessed on 29 March 2022)) [73], and the ML trees were constructed in IQ-TREE with 1000 non-parametric standard bootstraps performed to test the robustness of the clades [74].

Necropsy Findings, Bacterial Isolation, Histopathology, and RNAscope ® In Situ Hybridization Assay
Two animals exhibited swelling of the neck, and two other animals exhibit swelling of the tongue (Table 1, Figure 1A). Animal OV1300 showed diffuse, moderate petechia of the thoracic and abdominal fascia ( Figure 1B). Ten animals exhibited signs of pulmonary congestion and/or hemorrhage. One animal (OV1324) presented with severe suppurative pneumonia ( Figure 1C); cultures yielding both Gram-negative (Proteus sp., Pseudomonas sp.) and Gram-positive (Streptococcus sp.) bacteria. Escherichia coli and other members of the family Enterobacteriaceae were isolated from the lungs of 3/12 animals ( Table 2). The most common cardiac lesion was hemorrhage (9/12), ranging from mild petechia (6/12) to large, multifocal ecchymoses (1/12) ( Figure 1D). Congestion of the liver (8/12) and spleen (7/12) was commonly observed ( Figure 1E, Table 2).   Histopathology was performed on tissues from two animals (OV1248 and OV1296). Both animals exhibited mild to moderate hepatic congestion, and variable pulmonary congestion with edema. Animal OV1248 exhibited multifocal, acute, moderate, coagulative necrosis of tubular epithelial cells with associated interstitial hemorrhage and congestion in the kidneys (Figure 2A,B). The interalveolar septa were often infiltrated by low numbers of neutrophils, and small to medium caliber pulmonary vessels occasionally contained medium numbers of intraluminal and marginated neutrophils ( Figure 2C). Additionally, the lungs of animal OV1296 exhibited acute, multifocal, moderate hemorrhage ( Figure 2D,E) and moderate, multifocal, submucosal hemorrhage of the bronchi. In the heart, there was moderate, multifocal, subepicardial hemorrhage, elevating the epicardium and extending into the myocardium ( Figure 2F). The spleen exhibited moderate, multifocal hemorrhage. bers of neutrophils, and small to medium caliber pulmonary vessels occasionally contained medium numbers of intraluminal and marginated neutrophils ( Figure 2C). Additionally, the lungs of animal OV1296 exhibited acute, multifocal, moderate hemorrhage ( Figure 2D,E) and moderate, multifocal, submucosal hemorrhage of the bronchi. In the heart, there was moderate, multifocal, subepicardial hemorrhage, elevating the epicardium and extending into the myocardium ( Figure 2F). The spleen exhibited moderate, multifocal hemorrhage.  Heart, kidney, liver, and lung tissue sections from animals OV1248 and OV1296 were evaluated for EHDV-6 RNA using RNAScope ® in situ hybridization. EHDV-6 RNA was detected within scattered cells of the heart, kidney, liver, and lung ( Figure 3). Distribution of EHDV-6 labeling was most prominent in endothelial cells, especially in renal glomerular capillaries (Figure 3(A1)), alveolar capillaries of the lung (Figure 3(B1)), small myocardial vessels (Figure 3(C1)), and endothelial lining of the hepatic sinusoids (Figure 3(D1)). EHDV-6 labeling was occasionally observed within alveolar macrophages. The CytB probe (internal positive control) displayed labeling in the evaluated tissues (Figure 3(A2,B2,C2,D2)). No staining was observed when the dapB (negative control) probe was applied to the tissue sections (Figure 3(A3,B3,C3,D3)).   3.2. RT-PCR Detection of BTV, EEEV, EHDV, and WNV vRNA RNA extracts from one or more tissues from all 12 animals were positive for EHDV by RT-qPCR (Table 3). Spleen RNA extracts from six animals were positive for EHDV-6, one deer was positive for EHDV-2, and one was positive for both EHDV-2 and EHDV-6. Four animals yielded inconclusive results using the EHDV typing RT-PCR assay and were classified as "untypeable". The serotype of sample OV1288 was determined using the next-generation sequencing data (described below in Section 3.4), and it was classified as EHDV-6 based on its VP2 and VP5 gene sequences. All spleen vRNA extracts were negative for EEEV and WNV. The only animal positive for BTV was OV1299.

Virus Isolation
After 5 days post-inoculation, formation of cytoplasmic inclusions concomitant with granulation of the cytoplasm, evolving to enlargement of cells, and subsequent detachment from the growing surface were observed for samples OV1224, OV1248, OV1265, OV1288, OV1289, OV1296, OV1300, OV1314, and OV1321. No CPE was observed for samples OV1317 and OV1324 after two passages and they were considered negative for virus isolation.

Discussion
EHDV serotypes 1, 2, and 6 are endemic in farmed and free-ranging WTD populations in North America [21,22]. Our study confirmed an outbreak of an EHDV-6 strain in farmed WTD in Florida in 2019 based on clinical signs, gross and microscopic pathology, in situ hybridization (ISH), RT-PCR and RT-qPCR, virus isolation, and next-generation sequencing. Phylogenetic and genetic analyses of the coding sequences of six Florida EHDV isolates supported them as a novel U.S. reassortant EHDV-6 strain (OV1321).  Supplemental  Table S1.

Discussion
EHDV serotypes 1, 2, and 6 are endemic in farmed and free-ranging WTD populations in North America [21,23]. Our study confirmed an outbreak of an EHDV-6 strain in farmed WTD in Florida in 2019 based on clinical signs, gross and microscopic pathology, in situ hybridization (ISH), RT-PCR and RT-qPCR, virus isolation, and next-generation sequencing.
Phylogenetic and genetic analyses of the coding sequences of six Florida EHDV isolates supported them as a novel U.S. reassortant EHDV-6 strain (OV1321).
The gross and microscopic lesions associated with the Florida EHDV-6 outbreak in farmed WTD in 2019 were similar to those previously described for an experimental infection of EHDV-6 in WTD [26] including: (1) widespread hemorrhages on the abdominal muscle fascia, epicardium, and rumen wall; (2) mild to moderate hepatic congestion; (3) varying levels of pulmonary congestion, hemorrhage, and edema; (4) moderate subepicardial hemorrhage; and (5) moderate coagulative necrosis with associated interstitial hemorrhage and congestion of the kidneys. The EHDV-6 ISH labeling appeared subjectively greatest in cells morphologically resembling macrophages in the lung and liver parenchyma and the endothelium of all organs evaluated. These results are consistent with the monocytotropic and endotheliotropic nature of EHDV [26,75,76]. The prominent labeling of endothelium likely explains the vascular compromise and resulting multisystemic hemorrhage, pulmonary edema, and renal tubular coagulative necrosis. Similar to the intense staining of the lung sections by ISH, the RT-qPCR data supported high EHDV loads in the lungs (Ct values of 22 and 25 in OV1248 and OV1296, respectively). Taken together, the observed pathological findings and the results of the virus culture and molecular assays supported EHDV-6 as the cause of disease in the farmed WTD.
Genetic and phylogenetic analyses of the majority of the structural and non-structural genes supported the close relationship of the U.S. reassortant EHDV-6 strain (OV1321) to the Trinidad reassortant EHDV-6 strain and confirms the progenitor of these Western Hemisphere EHDV-6 strains to be an Australian EHDV-6 strain (CSIRO 753). The closely related U.S. reassortant EHDV-6 strain (OV1321) and the Trinidad reassortant EHDV-6 strain are unique among EHDV-6 strains in that they both received segment 4 from an Australian EHDV-2 strain (CSIRO 439) [63]. The U.S. reassortant EHDV-6 strain (OV1321) and the Trinidad reassortant EHDV-6 strain can be differentiated from each other in that the former possesses an NS2 gene derived from an Australian EHDV-8 strain (CPR 3961A) and the latter received its NS2 gene after it arrived in the Western Hemisphere from a yet unknown EHDV strain [63].
Previous phylogenetic analyses of the U.S. endemic reassortant EHDV-6 strain (Indiana) from diseased WTD in the central and eastern U.S. since 2006 revealed it evolved through reassortment with EHDV-6 strains from Australian cattle and a EHDV-2 strain from U.S. WTD [16,22,63]. The serotype determining outer capsid proteins (VP2 and VP5) of the U.S. endemic reassortant strain (Indiana) were derived from an Australian prototype EHDV-6 strain (CSIRO 753) and the remaining gene segments encoding both nonstructural and structural proteins derived from a U.S. EHDV-2 strain [16,22,63]. However, the donor of the two EHDV-6 segments in the U.S. endemic reassortant strain (Indiana) remained obscure [22]. Later studies suggested a reassortant EHDV-6 strain isolated from asymptomatic cattle in Trinidad, with 9/10 segments derived from Australian EHDV-6 (CSIRO 753) and EHDV-2 (CSIRO 439) strains, may have spread to the U.S. prior to 2006, where it then served as the donor of the EHDV-6 segments in the creation of the U.S. endemic reassortant EHDV-6 strain (Indiana) [16,63]. Previous phylodynamic analyses estimated that a prototype Australian EHDV-6 strain entered the Western Hemisphere around 1966 [16]. Additional evidence that the EHDV-6 serotype was circulating in the U.S. prior to 2006 is suggested by the detection of neutralizing antibodies against EHDV-6 in WTD in the southeastern U.S. as early as 2000 [77].
It is also possible that the U.S. novel reassortant EHDV-6 strain (OV1321) characterized in our study was present in the U.S. prior to 2006 and it served as the donor of the EHDV-6 segments to the U.S. endemic reassortant EHDV-6 strain (Indiana). However, our phylogenetic analyses, based on the VP2 and VP5 genes, did not resolve the relationship of the U.S. novel reassortant EHDV-6 strain (OV1321) to the other EHDV-6 strains (e.g., U.S. endemic, Australian, and Caribbean EHDV-6 strains). If the U.S. novel reassortant EHDV-6 strain (OV1321) has been present in the U.S. for some time, it is also possible that it spread from the U.S. to the Caribbean via movement of an infected ungulate host (such as cattle) or competent vector [16,63]. Additional complete genome sequences of EHDV-6 strains across a greater range of hosts (e.g., bovids/cervids), years (e.g., prior to 2006), and geographic regions (e.g., U.S., Caribbean, and South America) would facilitate phylodynamic modeling that could result in a better understanding of the timing and number of incursions of EHDV-6 strains into the Western Hemisphere, and their subsequent dissemination within the region.
The EHDV-6 seroprevalence is high in domestic cattle ≤ 2 years of age in Florida [78]. To date, EHDV-6 strains derived from the U.S., Caribbean, and Australia have not resulted in clinical disease in cattle [16,26,62,64]. However, the disease potential of the U.S. novel reassortant EHDV-6 strain (OV1321) in cattle remains to be determined. Future research is needed to determine the susceptibility and potential role in disease of this novel EHDV-6 strain in both domestic and wild ungulates.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14051012/s1, Figure S1: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 stain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the VP3 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Figure S2: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 strain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the VP6 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Figure  S3: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 strain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the NS1 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Figure S4: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 strain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the NS3 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Figure S5: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 strain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the VP5 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Figure S6: Maximum Likelihood cladogram depicting the relationships of the U.S. novel reassortant EHDV-6 strain isolated from a white-tailed deer (OV1321) to 66 other EHDV strains. The tree was generated from the nucleotide sequence alignment of the VP7 gene. The included EHDV and BTV strains are indicated by serotype, country, state (for U.S. strains), strain or isolate name, and GenBank accession no. Nodes with black circles are supported by bootstrap values ≥ 80%. The tree was rooted with the BTV-28 strain. Additional metadata for each virus in the tree are provided in Supplemental Table S1; Table S1: Serotype, strain/isolate name, country, U.S. state, year of detection, host, and GenBank accession numbers for all 10 segments of selected EHDV and BTV strains used in the phylogenetic analysis.