Peripheral transcriptional responses to experimental SARS-CoV-2 inoculation in North American Elk cows and calves

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Sarlo Davila, Alexandra C. Buckley, Eric D. Cassmann, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6505643/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Aug, 2025 Read the published version in BMC Genomics → Version 1 posted 12 You are reading this latest preprint version Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a health risk for humans and other domestic and wildlife species. Recently, North American elk has been identified as seropositive for SARS-CoV-2, thus posing a potential threat to humans and other mammals. In this work, we characterized the peripheral transcriptomic response to experimental SARS-CoV-2 infection in calves and adult elk. Results Significantly differentially expressed genes were identified at 2, 5, and 14 days post inoculation (dpi) for both age groups. Adult elk presented the greatest number of differentially expressed (DE) genes at all time points, including many genes associated with the viral response, immune activation, and antibody production, those associated with coronavirus disease (COVID-19), and coronavirus GO terms and KEGG pathways. Calves presented DE genes associated with viral responses at 5 dpi as well as neurodegenerative-associated genes at 14 dpi. Both adults and calves showed predicted activation of the ISGF3 and IFN type I pathways at 2 dpi and, globally, increased activity related to the coronavirus pathway disease at 5 and 14 dpi. Conclusions Collectively, this work provides valuable data characterizing the immune response of elk to viral diseases as well as the response of wildlife to SARS-CoV-2 infection. SARS-CoV-2 coronavirus RNA-seq zoonotic elk Cervus elaphus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), remains a public health concern and one of the most severe global public health emergencies in history, killing more than 7 million people around the world (World Health Organization - https://data.who.int/dashboards/covid19/deaths ) by March 2025. In addition to its impact on human health, SARS-CoV-2 has also been a concern in various domestic, peridomestic, and wild animals, including cats, dogs, mice, rats, American mink ( Neovison vison ), Eurasian river otters ( Lutra lutra ), ferrets ( Mustela furo ), Syrian hamsters ( Mesocricetus auratus ), gorillas, lions, tigers, Virginia opossums ( Didelphis virginiana ), raccoons ( Procyon lotor ), groundhog ( Marmota monax ), red bats ( Lasiurus borealis ), white-tailed deer ( Odocoileus virginianus ), red deer ( Cervus elaphus ), and fallow deer ( Dama dama ) [ 1 , 2 ]. At the molecular level, the most defining structural protein of SARS-CoV-2 is the spike (S) protein. During infection, the S protein binds to surface angiotensin-converting enzyme 2 (ACE2), the receptor responsible for virus entrance into the cell, facilitating virus replication [ 3 , 4 ]. The variability in the specificity of the interaction between viruses and receptors is believed to reflect the range of susceptible hosts. The ACE2 protein of humans has a high degree of homology with that of white-tailed deer, reindeer, and Pierre Davids’s deer [ 5 ]. White-tailed deer were previously shown to be highly susceptible to infection with viral shedding and transmission to other white-tailed deer [ 5 , 6 ]. Subsequent surveys of wild white-tailed deer across the United States (US) revealed widespread exposure/infection, which suggests ongoing deer-to-deer transmission and a possible source of deer-to-human infection. Although white-tailed deer are the most abundant wild ungulate in North America, with approximately 30 million found in the US alone, another closely related but less numerous large ungulate is North American elk ( Cervus elaphus canadensis ). In a recent publication [ 7 ] from our own laboratory, the susceptibility of calves and adult elk to SARS-CoV-2 infection was investigated. Although elk are moderately permissive to SARS-CoV-2 infection and exhibit seroconversion, viral shedding and tissue distribution are much lower than those observed in white-tailed deer. Interestingly, viral RNA and viral protein were detectable in lymphoid tissues 21 days after infection in both elk calves and adults. Based on these findings and to further understand virus‒host interactions, we investigated the changes in whole-blood gene expression in elk calves and adults following experimental challenge with SARS-CoV-2. Understanding the transcriptomic response to infection could shed light on SARS-CoV-2 pathophysiology in mammals, including age-related differences, which have not been investigated previously. Results We evaluated the RNA-seq whole-blood transcriptomes of calves and adult elk cows experimentally infected with USA-WA1/2020 SARS-CoV-2, on days 0, 2, 5, and 14 post inoculation (pi). Differentially expressed genes between day 0 and days 2, 5, and 14 post-SARS-CoV-2 inoculation in elk calves In elk calves, the comparison of day 2 pi to day 0 revealed 715 genes that were significantly differentially expressed (DE), with 183 downregulated genes and 532 upregulated genes (p-adjusted < 0.05) (Fig. 1 a, see Additional file 1). Among the upregulated genes, 24 were involved in the coronavirus disease pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway) (Additional file 2). Interestingly, upregulated genes in this pathway included regulators of a “cytokine storm” immune response to SARS-CoV-2, such as CXCL10 (log2fc = 2.60) [ 8 ], IRF3 (interferon regulatory factor 3, log2fc = 0.36), IRF9 (interferon regulatory factor 9, log2fc = 1.00), MX1 (MX Dynamin Like GTPase 1, log2fc = 3.70) and MX2 (MX Dynamin Like GTPase 2, log2fc = 4.70). Both MX1 and MX2 are linked to the cellular antiviral immune response [ 9 ]. In addition, ISG15 (ISG15 ubiquitin-like modifier, log2fc = 3.90) was upregulated and has previously been implicated in inflammatory responses after SARS-CoV-2 exposure [ 10 ]. Compared with day 0, the day 5 pi analysis revealed 921 DE genes; 339 were downregulated, and 582 were upregulated (Fig. 1 b, see Additional file 3). Several upregulated genes were associated with immune response pathways associated with viral responses, including the NOD-like receptor signaling pathway (20 genes), herpes simplex virus 1 infection (20 genes), Epstein‒Barr virus infection (19 genes), hepatitis C (17 genes), influenza A (17 genes), human papillomavirus infection (16 genes), and the biological term for defense response to virus (12 genes) (see Additional file 4). A comparison of day 14 pi versus day 0 revealed the greatest number of DEGs, with a total of 7,179 DEG, of which 3,602 were downregulated and 3,577 upregulated (Fig. 1 c, see Additional file 5). Some of the upregulated genes were associated with known SARS-CoV-2 players, such as IRAK1 (interleukin 1 receptor-associated kinase 1, log2fc = 0.87), IRF3 (interferon regulatory factor 3, log2fc = 0.59), ISG15 (ISG15 ubiquitin-like modifier, log2fc = 1.30) and IRF9 (interferon regulatory factor 9, log2fc = 0.66). Additionally, upregulated genes were associated with protein binding, ATP binding, structural constituents of ribosomes, and translation GO terms, as well as thermogenesis and ribosomes as KEGG pathways, all of which are associated with viral responses. Interestingly, several upregulated DEGs were related to neurodegenerative diseases, such as genes related to Alzheimer’s disease: PSEN2 , TREM2 , and GAB2 [ 11 – 13 ]; Parkinson’s disease: ATP13A2 and DCTN1 [ 14 ]; and amyotrophic lateral sclerosis: TAF15 and FUS [ 15 , 16 ] (see Additional file 6). Differentially expressed genes between day 0 and days 2, 5, and 14 post SARS-CoV-2 inoculation in adult elk Compared with those of day 0 samples, the DEG analysis of day 2 pi samples revealed 2,756 DE genes (see Additional file 7). In total, 1,421 genes were upregulated, whereas 1,234 genes were downregulated (p adjusted < 0.05) (Fig. 2 a). The upregulated genes were associated with coronavirus disease KEGG pathways, including members of the MAPK family of cascades, such as MAPK3 (mitogen-activated protein kinase 3) and MAPKAPK3 (MAPK activated protein kinase 3) (with log2-fold change values = 0.70 and 0.47, respectively); Myd88 (myeloid differentiation primary response gene, log2fc = 0.57); IRF3 (interferon regulatory factor 3, log2fc = 0.84); ISG15 (ISG15 ubiquitin-like modifier, log2fc = 4.94); STAT1 (signal transducer and activator of transcription 1, log2fc = 0.99); and STAT2 (signal transducer and activator of transcription 2, log2fc = 1.00). Most of these genes are involved in various immune system pathways, including antiviral defense (see Additional file 8). Analysis revealed 9,708 DE genes in the adult elk between day 5 pi and day 0; of these genes, 5,399 were downregulated, and 4,309 genes were upregulated (p-adjusted < 0.05) (Fig. 2 b, see Additional file 9). Several genes presented very large log2-fold changes in both directions (up- and downregulated). Examples include VIM (vimentin, log2fc = -17.77), ADA (adenosine deaminase, log2fc = -16.47), TRIM15 (tripartite motif containing 15, log2fc = 17.82), FAM227A (family with sequence similarity 227 Member A, log2fc = 15.70) and MAJIN (membrane anchored junction protein, log2fc = 15.29). Extended results can be found in Additional file 10. Among the genes upregulated for this comparison, 106 were associated with the coronavirus disease KEGG pathway and included ACE2 (angiotensin-converting enzyme 2, log2fc = 2.87), TNF (tumor necrosis factor, log2fc = 3.46), and MAPK family members, such as MAPK4 (mitogen-activated protein kinase 4, log2fc = 9.34), MAPK11 (mitogen-activated protein kinase 11, log2fc = 10.06), and ISG15 (ISG15 ubiquitin-like modifier, log2fc = 3.21). Between day 14 pi and day 0, 3,950 genes were DE; 1,799 were downregulated, and 2,151 were upregulated, with a p-adjusted < 0.05 (Fig. 2 c, see Additional file 11). As observed on day 5 pi, there were also large log2-fold changes in ALAS2 (5'-Aminolevulinate Synthase 2, log2fc = -19.11), FTH1 (Ferritin Heavy Chain 1, log2fc = -19.03), GNAI2 (G Protein Subunit Alpha I2, log2fc = -18.78), UCP3 (Uncoupling Protein 3, log2fc = 16.43), MAJIN (Membrane Anchored Junction Protein, log2fc = 15.04) and H1-1 (H1.1 Linker Histone, Cluster Member, log2fc = 14.69). KEGG pathway analysis revealed 101 upregulated DEGs associated with the coronavirus disease - COVID-19 KEGG pathway, including MAS1 (MAS1 Proto-Oncogene, G Protein-Coupled Receptor, log2fc = 10.46), TNF (Tumor necrosis factor, log2fc = 4.82), MAPK family members such as MAPK11 (Mitogen-Activated Protein Kinase 11, log2fc = 7.10) and MAPK12 (Mitogen-Activated Protein Kinase 12, log2fc = 4.69). Genes such as UCP3 (uncoupling protein 3), MAJIN (membrane anchored junction protein), H1-1 (H1.1 linker histone, cluster member) and HAS1 (hyaluronan synthase 1) presented log2fc values of 16.43, 15.04, 14.69 and 14.32, respectively. ISG15 was also upregulated, with log2fc = 1.17 (see Additional file 12). Comparison of differentially expressed genes between elk calves and adults We also compared the overlap between significantly up- and down-regulated DE genes (Fig. 3 ), within timepoints between adults and calves to understand the similarities and differences in the immune response. When the downregulated genes between the adult and calf groups on day 2 pi vs. day 0 were compared, 35 genes overlapped. Most of them associated with the Golgi apparatus, including STK26 (serine/threonine-protein kinase 26) and HOOK3 (hook microtubule tethering protein 3) but also include GEMIN6 (gem nuclear organelle-associated protein 6), a gene already associated with SARS-CoV-2 infection [ 17 ] (Fig. 3 a). Among the 239 upregulated genes shared between adults and calves, 19 genes were related with the coronavirus disease-COVID-19 KEGG pathway, and the others were associated with additional antiviral responses, including herpes simplex virus 1 infection, influenza A, the defense response to a virus, hepatitis C, and NOD-like receptor signaling pathways. When DE gene expression from both ages of elk was compared between day 5 pi and day 0 (Fig. 3 b), 170 downregulated genes were shared. Many of these genes had GO terms related to the nucleus, regulation of transcription by RNA polymerase II, DNA-binding transcription factor activity, RNA polymerase II-specific and ATP binding. The associated KEGG pathways included the regulation of the actin cytoskeleton, cellular senescence and the MAPK signaling pathway. On the upregulated side, 164 genes were shared between adults and calves, 10 of which were associated with the KEGG pathways for coronavirus disease-2019 (COVID-19), other genes related to the NOD-like receptor signaling pathway, and different viral infections, such as influenza A, hepatitis C, Epstein–Barr virus infection, and herpes simplex virus 1 infection. A comparison of the adult and calf DE gene lists between day 14 pi and day 0 (Fig. 3 c) revealed that 2,241 downregulated genes were shared. The genes with reduced expression were associated with KEGG pathways related to viral infections, including herpes simplex virus 1 disease, human papillomavirus infection, and human cytomegalovirus infection, and with GO terms associated with the nucleus, protein binding, cytoplasm, ATP binding and regulation of transcription by RNA polymerase II. Among the 1,043 shared upregulated DE genes, more than 100 genes were associated with metabolic KEGG pathways, and many of them had GO terms related to the cytoplasm, cytosol and mitochondrion. Additionally, 34 genes were involved in pathways associated with neurodegeneration, including multiple diseases, such as 29 genes associated with Alzheimer’s disease, 28 genes associated with Huntington’s disease, and 26 genes associated with prion disease and Parkinson’s disease. Finally, we determined which DE genes presented up- and downregulated expression levels across both groups and all timepoints. In total, 7 genes were found to be upregulated in both groups across all time points: G6PC3, IFI35, LOC122430223, IRF9, LGALS9, DHX58 and ISG15 . In the downregulated group, 6 DE genes overlapped between adults and calves at different timepoints: STK26 , EDEM3 , ADSS2 , CPNE3, STRN and ADAM10 . Ingenuity Pathway Analysis of differentially expressed genes between adult elk and calves To further analyze the elk response to SARS-CoV-2, we used the Ingenuity Pathway Analysis (IPA) software to examine changes in the coronavirus pathogenesis pathway. We compared the predicted intracellular responses at different stages of infection over time and between calves (Fig. 4 ) and adult elk (Fig. 5 ). In calves, DE genes between day 2 pi and day 0 (Fig. 4 a) suggest activation of the innate immune system. The molecular activity predictor tool highlighted the upregulation of the signal transducers and activators of transcription (STAT) pathway, with the STAT1 and STAT2 genes activated, which are known mediators of antiviral host defense and interferon response signaling. Similarly, the terms IFN-1 and ISGF3 were activated, contributing to proinflammatory NF-kB and interferon type 1 responses. Figure 4 b shows the comparison of DE genes from day 5 pi and day 0, where the ISGF3 cascade remained activated in calves, along with STAT1 , STAT2, NF-kB and Interferon type 1 responses. The IPA predicted the inhibition of SARS-CoV-2 replication and continued activation of the innate and adaptive immune responses, including associations with pathway terms such as apoptosis and cytokine storms by hypercytokinaemia. A comparison of day 14 versus day 0 (Fig. 4 c) revealed broad activation of pathway terms related to infection: IFN-1 remained active, and SARS-CoV-2 replication was still predicted to be inhibited. Transforming growth factor (TGF)-beta receptor activity was inhibited, which was linked to the downregulation of the SMAD3 gene. Notably, in calves, the spike protein receptor ACE2, which is necessary for virus entry into the cell, was predicted to be activated only on day 14 DE gene list. In adult elk, inflammatory signals associated with infection were more broadly activated in the IPA analysis of DE genes between day 2 pi and day 0 (Fig. 5 a). The TGFB , AGTR4 STAT1 , STAT2 , and ISGF3 genes were upregulated, along with Interferon type 1, adaptive, and innate immune activation. Interestingly, in contrast to that in calves, the ACE2 receptor gene was predicted to be activated earlier in adult animals as compared to the calves. A comparison of the DE gene predictions for day 5 pi and day 0 revealed a marked decrease in the immune response in adult animals (Fig. 5 b). A ‘deactivation’ of Interferon type 1 response was seen, as well as a decrease in the involvement of STAT1 , STAT2, ISGF3 , TGFB and the AGTR4 receptor. By day 5 pi in adults, the ACE2 receptor and interferon type 1 response were still activated. However, in the DE gene lists from day 14 pi, the ACE2 receptor gene was no longer activated, and the interferon type 1 response was inhibited along with a decrease in the expression level of other inflammatory genes, such as NF -kB , STAT1 and ISGF3 (Fig. 5 c). Discussion Here, we characterize for the first time the changes in the peripheral blood gene expression profile of calves and adult elk experimentally challenged with SARS-CoV-2. Our data illustrates a broad and rapid immune response to SARS-CoV-2 inoculation. Gene expression profiles highlight the changing expression of genes related to important pathways for controlling viral infection and activating the host immune system response. These data offer an important resource for comparative studies in elk and other mammals, including humans, to help understand the molecular machinery behind the antiviral response. The differentially expressed gene analysis consistently revealed upregulated genes involved in SARS-CoV-2 infection processes and Coronavirus disease KEGG pathways in both calves and adult elk. In a recent publication from our group [ 7 ], both elk calves and adults are susceptible to infection and have the ability to develop neutralizing antibodies against an ancestral SARS-CoV-2 variant (USA-WA1/2020). Interestingly, however, calves had a higher neutralizing response compared to the adults. Additionally, viral RNA and viral protein persisted in the retropharyngeal lymph nodes of both adults and calves. Our results suggest that age may have a significant impact on the elk response to SARS-CoV-2 challenge. The literature defines significant age-dependent differences in the immune response reported in humans [ 18 , 19 ], where older individuals present more evidence of high morbidity and mortality than do younger individuals, who have a more effective immune response to spike and peptides against SARS-CoV-2. The severity of coronavirus disease (COVID-19) in adult humans could result from the decreased potential to immunomodulate excessive inflammation after strong cytokine secretion by T cells [ 20 ]. Consistent with those findings, in our study, the variation in the transcriptomic response between calves and adult elk was illustrated by the substantial increase in DE genes at all timepoints in the adult elk and the lack of conservation across DEGs from both ages. In the elk calves, a relatively small number of genes were DE; however, immune activation and response trends were evident. Within the day 2 pi comparison, the upregulated genes were associated with GO terms and KEGG pathways related to coronavirus disease, the defense response to virus, and influenza A and herpes simplex virus 1 infection. Viral activation was more emphasized for calves on day 5 pi, when there was a significant increase in the expression of genes related to an antiviral immune response, such as genes from the interferon regulatory factor (IFR) family, interferon gamma-inducible proteins and other interferon-stimulated genes (ISGs). Notably, in calves on day 14 pi, there was an increased number of DE upregulated genes associated with neurodegenerative disease KEGG pathways. While we did not observe any clinical signs associated with neurological disease in the calves, it is worth noting that in humans, neurological symptoms and neuroinflammatory events have been described during and following infection with SARS-CoV-2 [ 21 , 22 ]. In adult elk, many DE genes were related to coronavirus disease - COVID-19 KEGG pathway and participating in immune system activation and the defense response to this coronavirus and other viral diseases. The GO and KEGG terms associated with COVID-19 appeared as early as day 2 pi. By day 5 pi, we observed larger log2-fold changes in expression, and genes related again to coronavirus disease – COVID-19 KEGG pathway. Some of the genes with larger log2-fold change are important to the viral and bacterial pathogens infections. VIM (Vimentin) have been shown to facilitate the binding of pathogens to the cell surface and promote their persistence in the host cells [ 23 ], and TRIM15 is a member of the TRIM family, a new class of antiviral proteins and essential components for innate antiviral immunity [ 24 ]. The impact of infection on day 14 pi is still evident, with log2-fold changes of approximately + 16 for upregulated genes and − 19 for downregulated genes, which were still associated with coronavirus disease pathways. This further highlight possible differences in immune responses, including the longevity of infection, between young and adult individuals across species. Significantly DE genes conserved between calves and adults could be potential biomarkers of SARS-CoV-2 infection in wildlife and/or other hosts. Across calves and adult elk, seven upregulated DEGs were shared across all time points: G6PC3, IFI35, LOC122430223, IRF9, LGALS9, DHX58 and ISG15 . Glucose-6-phosphatase catalytic subunit 3 (G6PC3) is associated with macrophage activation, regulation of signal transduction, positive regulation of the defense immune response, and innate immune system activation [ 25 ]. Interferon-induced protein 35 ( IFI35 ) is involved in the type I interferon signaling pathway and has been shown to be upregulated in patients with mild clinical COVID-19 [ 26 ]. Interferon regulatory factor 9 ( IRF9 ) is an important component of the type I and III interferon signaling pathways, and it has been associated with STAT1 and STAT2—also significantly DE genes in our study—to form the trimeric transcription factor ISGF-3 , which is associated with cellular antiviral responses [ 27 ]. The galectin-9 gene ( LGALS9 ) is a beta-galactoside-binding protein that is involved in cell signaling, adhesion, and migration and is involved in inflammatory processes [ 28 ]. The LGALS9 protein has already been described as a potential biomarker for many diseases, including COVID-19, and is highly expressed in hospitalized patients [ 29 ]. Another study revealed that this gene potentially enhances virus replication and inflammation in epithelial cells in vitro , suggesting that galectin-9 impacts the early stage of SARS-CoV-2 infection [ 30 ]. The DExH-box helicase 58 ( DHX58 ) gene is a member of a large family of RNA helicases and is responsible for encoding the LGP2 protein, which is important for RNA metabolism, splicing, recognition, modification, degradation, transport, and translation [ 31 ]. LGP2 proteins are involved in immunity against viruses. A study using a transgenic mouse model overexpressing this protein revealed a significant reduction in inflammatory mediators and low leucocyte infiltration into the bronchoalveolar space, resulting in a significant survival advantage compared with the wild-type group and leading to a reduction in the expression of the influenza A virus inflammatory response [ 32 ]. Like influenza A virus, SARS-CoV-2 results in infection of respiratory epithelial cells, so increased production of LGP2 genes could help reduce the incidence of clinical disease. The ISG15 gene was upregulated at all post challenge timepoints, with high log2-fold changes early after inoculation ( i.e. , 3.90 at 2 pi in calves and 4.94 at 2 pi in adults). As an IFN-stimulated gene (ISG), ISG15 is a key orchestrator of immune defense during viral infection and has already been described participating in the SARS-CoV-2 infection, inflammation, and immune responses [ 33 ]. ISG15 plays a vital role in cell cycle regulation, maintenance of genome stability, and innate immune signaling after viral infection [ 34 ]. An additional conserved gene that was DE in both calves and adults across all time points and has previously been reported in relation to SARS-CoV-2 infection was TNFAIP3 (tumor necrosis factor, alpha-induced protein 3). Sarlo-Davila et al. observed that the TNFAIP3 gene was upregulated in deer respiratory epithelial cells in vitro. In our study, TNFAIP3 was downregulated on day 5 (log2fc=-6.06) and day 14 (log2fc=-14.08) pi in adult elk, whereas the same gene was upregulated (log2fc = 0.40) on day 2 and downregulated (log2fc=-0.44) on day 14 pi in the calf group. It is known that some viruses can downregulate components of the immune system, including the receptor necessary for host cell entry, to prevent reinfection of the same cell [ 36 , 37 ]. Here, we also investigated the downregulated DE genes shared between calves and adult elk at different timepoints and identified six genes: STK26 , EDEM3 , ADSS2 , ADAM10 , CPNE3 and STRN . The STK26 (Serine/Threonine Kinase 26), also known as MST4, is a protein kinase that is expressed in monocytes and T, B and NK cells and mediates bacterial infections [ 38 ]. Compared with that in asymptomatic patients, the expression of STK26 is increased in sick human patients after SARS-CoV-2 infection [ 39 ]. A study investigating drug target proteins revealed that SARS-CoV-2 infection can change the structure of endoplasmic reticulum (ER) proteins at the initiation of virus‒cell interactions [ 40 ]. EDEM3 (ER Degradation Enhancing Alpha-mannosidase Like Protein 3) is a member of a group of proteins that perform glycoprotein degradation in the ER [ 41 ]; EDEM3 was downregulated in our study, indicating the possible interference of the virus in protein production. Adenylosuccinate synthase (ADSS) is known as a convertor of inosine monophosphate to adenosine monophosphate (AMP) and is present in vertebrates as two different isozymes: ADSS1 , which is the basic form that participates in the purine nucleotide cycle, and the acidic form ADSS2 , which catalyzes the synthesis of AMP [ 42 , 43 ]. Another downregulated gene shared between the groups is Copine 3 ( CPNE3 ), a member of the CNPE family; this gene is strongly expressed in the early stage of neutrophil differentiation [ 44 ] and plays an important role in maintaining the normal function of pancreatic β-cells, regulating glucose uptake and insulin secretion [ 45 ]. ADAM10 (ADAM metallopeptidase domain 10) is an adhesion receptor, and the mRNA that encodes this gene has been found in immune cells and is involved in the activation, differentiation and migration of T cells [ 46 ]. ADAM proteins, important membrane-anchored enzymes that play important roles in converting cytokine precursors near the membrane into soluble bioactive proteins, mediate the regulation of the post-translational function of proteins in the immune system [ 47 ]. The ADAM10 gene is also involved in the regulation of cytokine production and alternative protease cleavage, characterizing some important host dependency factors for SARS-CoV-2 entry into cells [ 48 ]. Ingenuity pathway analysis illustrated the participation of DE genes in the activation of adaptive and innate immune responses during SARS-CoV-2 infection. As soon as day 2 pi, both groups of elk had signatures consistent with innate and adaptive immune activation as well as the inhibition of the replication of SARS-CoV-2, which is consistent with most host approaches to control infection. Specifically, interferon-associated terms such as Interferon Stimulated Factor 3 ( ISGF3) , NF-kB, and general interferon type 1 responses were consistently activated. Type I interferons and associated mediators are known to play protective roles in controlling viral infections [ 49 ]. One of those genes is IRF3 (interferon regulatory factor 3), the primary early regulatory factor responsible for inducing IFN-1 during viral infection, which positively regulates apoptosis and inhibits NF-kB translocation [ 49 , 50 ]. IRF7 (interferon regulatory factor 7) can mediate inflammatory responses, and a lack of IRF9 results in increased COVID-19 risk [ 49 ]. Broadly, the host immune response, inflammatory, and antiviral signaling pathways were the highest early in the adult elk when the day 2 pi profile was examined, but they appeared to decline over time, as evidenced by the inhibition of immune terms by day 5 pi. In contrast, the calves appeared to show an increase in the immune response and interferon activation through day 5 pi and only moderately decreased in activity by day 14 di. Notably, adults showed a predicted involvement of ACE2 as early as day 2 and through day 5 pi, whereas calves showed activation of ACE2 only on day 14 pi. Both calves and adult elk have been shown to lack clinical signs associated with SARS-CoV-2 disease, suggesting that while elk can become infected, it is successful at controlling the infection without causing disease. Collectively, this work provides unique insight into potential mechanisms of viral control by the host immune system, variation in disease responses among age groups, and gene signature associations with stages of infection. Conclusions Here, for the first time, we describe the peripheral transcriptional response of North American elk to SARS-CoV-2 infection at different timepoints and in different age groups. Overall, this work facilitates the discussion of species-specific responses to a zoonotic pathogen with global human health implications, provides further characterization of wildlife immune responses, and highlights the gene expression profiles associated with SARS-CoV-2. Methods Elk challenge and sample collection All procedures and animal work were performed following approval by the NADC Animal and Care Use Committee (IACUC) and follow the Guide for Care and Use of Laboratory Animals regulations. For the challenge studies, adult elk (n = 10), approximately 4 years of age, were transferred to agricultural biosafety level 3 (AgBSL3) facilities and allowed to acclimate for 2 weeks. Elk calves (n = 11), approximately 5 months of age, were born in high containment, and remained with dams until weaning. All animals were challenged with an ancestral strain of SARS-CoV-2 (USA-WA1/2020), as described previously in Boggiatto et al. (2024). Briefly, the animals were challenged intranasally with a SARS-CoV-2 isolate (USA-WA1/2020 – SARS-Related Coronavirus 2, Isolates hCoV-19/USA-WA1/2020). Blood samples were collected from elk calves and elk adult cows pre-challenge day 0 (control), day 2, day 5, and day 14 post- inoculation using PAXgene® Blood RNA tubes (BD Biosciences, San Jose, CA). RNA isolation, library preparation and sequencing Total RNA was isolated using MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit, which is compatible with PAXgene™ Blood RNA Tubes (catalog number 4451894) (Thermo Fisher Scientific, Waltham, MA). The RNA integrity was determined via a bioanalyzer, and the average RNA integrity number (RIN) for all the samples was 9.3. Library preprocessing was performed via an Illumina mRNA kit, and sequencing was performed at the University of Illinois, Urbana-Champaign, at Roy J. Carver Biotechnology Center via Nova Seq X Plus, which generated 150 base pair paired-end reads. RNA sequencing bioinformatics: quality control and read mapping Data processing was performed using the Nextflow (version 24.04.2) nf-core/rna-seq pipeline (version 3.14.0) [ 51 , 52 ]. Read quality control (QC) was processed by FastQC software ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ , version 0.12.1), and the library adaptors were trimmed using TRIMgalore software ( https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ , version 0.6.7), removing the reads with Phred quality scores lower than 20. After filtering, the sequence reads were aligned against the Cervus canadensis genome assembly (ASM19320065v1) via STAR software [53, version 2.7.10a]. Gene counts were performed with featureCounts software [54, release 2.0.4], and all subsequent statistical analyses were performed in the R environment. Differentially expressed gene analysis The differentially expressed (DE) analysis was performed comparing each challenged day post-inoculation (day 2, day 5 and day 14) to the control group (day 0) for both the adult and calf groups, using DESeq2 software in R [ 55 ], version 1.44.0. For the DE analysis, reads were filtered to remove genes with no expression (zero reads), genes with very low expression (less than 1 read per sample on average), and genes with rare expression across the samples (genes with read counts that were not present in at least 3 samples). GO term enrichment analysis and Ingenuity Pathway Analysis Gene Ontology (GO) enrichment analysis and KEGG pathway analysis were performed using DAVID bioinformatics software ( https://david.ncifcrf.gov/tools.jsp ) to help interpret and summarize the biological processes and pathways associated with the DE genes. The overlap between DE genes in the different groups was determined via Venny [56, version 2.1]. Ingenuity pathway analysis (IPA) (QIAGEN, Redwood City, CA, USA) [ 57 ] was performed using the gene expression data as input to predict the activation or inhibition of the metabolic canonical pathways in a core analysis. Declarations Ethics approval All procedures and animal work were performed following approval by the NADC Animal and Care Use Committee (IACUC) and follow the Guide for Care and Use of Laboratory Animals regulations. Data availability The dataset supporting the conclusions of this article is available in the SRA-NCBI repository under project number PRJNA1196870 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1196870). Competing interests The authors declare no competing interests . Funding This work was funded by intramural funding from the U.S. Department of Agriculture and American Rescue Plan (ARP) funds through an interagency agreement between the Agricultural Research Service (ARS) and the Animal and Plant Health Inspection Service (APHIS) Wildlife Service (WS). Additionally, this research was supported in part by an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE-SC0014664. All the opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of the USDA, DOE, or ORAU/ORISE. Author contributions B.P. analyzed data and wrote the manuscript. K.M.S.D. analyzed data and reviewed the manuscript. A.C.B. participated in study design, prepared the virus for infections, assisted with animal work, sample collection, and sample processing. E.D.C. participated in study design, collected samples. S.C.O. performed animal experiments and collected samples. P.M.B. participated in study design, performed the animal experiments, collected samples, analyzed data, and reviewed the manuscript. M.V.P. obtained funding, managed project, participated in study design, performed animal experiments, and collected samples. E.J.P. participated in the study design, processed samples, analyzed data, and reviewed the manuscript. Acknowledgments The authors would like to thank the National Animal Disease Center (NADC) Animal Resources Unit (ARU) for the expert care of the animals used in this study. Specifically, the authors thank Dr. Rebecca Cox, Derek Vermeer, Jonathan Gardner, Tiffany Williams and Kolby Stallman for their animal husbandry, care, and assistance. 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Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21. Oliveros JC. Venny. An Interactive Tool for Comparing Lists with Venn’s Diagrams. 2015. Krämer A, Green J, Pollard J, Tugendreich S. Causal analysis approaches in ingenuity pathway analysis. Bioinformatics. 2014;30:523–30. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Additional file 1. (Additional file 1.xlsx) List of genes upregulatedand downregulated in elk calves 2 days after SARS-CoV-2 challenge. Additionalfile2.xlsx Additional file 2. (Additional file 2.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 2 days after SARS-CoV-2 challenge. Additionalfile3.xlsx Additional file 3. (Additional file 3.xlsx) List of genes upregulatedand downregulated in elk calves onday 5 after SARS-CoV-2 challenge. Additionalfile4.xlsx Additional file 4. (Additional file 4.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 5 days after SARS-CoV-2 challenge. Additionalfile5.xlsx Additional file 5. (Additional file 5.xlsx) List of genes upregulated and downregulated in elk calves on day 14 after SARS-CoV-2 challenge. Additionalfile6.xlsx Additional file 6. (Additional file 6.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 14 days after SARS-CoV-2 challenge. Additionalfile7.xlsx Additional file 7. (Additional file 7.xlsx) List of genes upregulatedand downregulated in adult elk on day 2 after SARS-CoV-2 challenge. Additionalfile8.xlsx Additional file 8. (Additional file 8.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 2 days after SARS-CoV-2 challenge. Additionalfile9.xlsx Additional file 9. (Additional file 9.xlsx) List of genes upregulatedand downregulated in adult elk on day 5 after SARS-CoV-2 challenge. Additionalfile10.xlsx Additional file 10. (Additional file 10.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 5 days after SARS-CoV-2 challenge. Additionalfile11.xlsx Additional file 11. (Additional file 11.xlsx) List of genes upregulated and downregulated in adult elk on day 14 after SARS-CoV-2 challenge. Additionalfile12.xlsx Additional file 12. (Additional file 12.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 14 days after SARS-CoV-2 challenge. Cite Share Download PDF Status: Published Journal Publication published 28 Aug, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 27 May, 2025 Reviews received at journal 25 May, 2025 Reviews received at journal 21 May, 2025 Reviews received at journal 19 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 12 May, 2025 Editor invited by journal 28 Apr, 2025 Editor assigned by journal 24 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 22 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Figure 1a shows the DEGs onday 2 post inoculation (pi) compared with those on day 0. Figure 1b shows the DEGs when day 5 pi was compared with day 0. Figure 1c shows the DEGs on day 14 pi compared with those on control day 0. The red dots representthe DE genes whoselog2-fold change was greater than 1.0 on both sides (upregulated and downregulated) and whose adjusted p value was \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/a6ec1bb29f3fabda517684e8.png"},{"id":82784765,"identity":"be4d44eb-c050-4e5d-b61e-9c0ae5d4c625","added_by":"auto","created_at":"2025-05-15 08:57:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":792137,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot representing genes whoselog2-fold change was greater than 1.0 and significant (p adjusted \u0026lt; 0.05) in the DE gene analysis ofadult elk. Figure 1a shows the DEGs between day 2 post-inoculation (pi) compared to day 0. Figure 1b shows the DEGs between day 5 pi compared to day 0. Figure 1c showsthe DEGs betweenday 14 pi compared to day 0. The red dots representthe DE genes whoselog2-fold change was greater than 1.0 on both sides (upregulated and downregulated) and whose adjusted p value was \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/b5f3191f105aa3c1603b7c5e.png"},{"id":82785118,"identity":"a1fd9c54-d977-453d-9c35-be0e577068fc","added_by":"auto","created_at":"2025-05-15 09:05:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1104838,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed (DE) genes overlapping between the calfand adult elk groups, separated by downregulated and upregulatedgenes. Figure 4a. DE genes on day 2 post-inoculation (pi) in calves and adult elk. Figure 4b. DE genes on day 5 pi in calves and adult elk. Figure 4c. DE genes on day 14pi in calves and adult elk.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/b0bfd3470e1ad3a973913e4f.png"},{"id":82785119,"identity":"2f2fc59b-43a0-4477-8157-b4ab3b31ec39","added_by":"auto","created_at":"2025-05-15 09:05:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4053676,"visible":true,"origin":"","legend":"\u003cp\u003eIngenuity pathway analysisshowing the differentially expressed (DE) genes involved in the coronavirus pathogenesis pathway inelk calves. The predicted activated and inhibited pathway associations for the DE gene lists are shown for day 0 vs day 2 (Figure 4a), day 5 (Figure 4b), and day 14 (Figure 4c) post-SARS-CoV-2 infection. Downregulated genes are shown ingreen, and upregulated genes are shown inpink. The Molecule Activity Predictor tool was used to predict downstream activity on the basis of significant differential gene expression. Predicted activation is shown in orange, and predicted inhibition is shown in blue. The darker the fill is, the more confident the prediction. The solid lines represent direct relationships, whereas thedashed lines represent indirect relationships.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/bd2b63e48d6e0ac9aa955837.png"},{"id":82784779,"identity":"02e63b7b-3215-4e8f-8832-3bfd29ae3420","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4770933,"visible":true,"origin":"","legend":"\u003cp\u003eIngenuity pathway analysisshowing the differentially expressed (DE) genes involved in the coronavirus pathogenesis pathway inadult elk. The predicted activated and inhibited pathway associations for the DE gene lists are shown for day 0 vs day 2 (Figure 5a), day 5 (Figure 5b), and day 14 (Figure 5c) post-SARS-CoV-2 infection. Downregulated genes are shown ingreen, and upregulated genes are shown inpink. The molecule activity predictor tool was used to predict downstream activity based on significant differential gene expression. Predicted activation is shown in orange, and predicted inhibition is shown in blue. The darker the fill is, the more confident the prediction. The solid lines represent direct relationships, whereas thedashed lines represent indirect relationships.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/803b8fd438355d234c1a5784.png"},{"id":90344956,"identity":"59003f6a-7cd2-43bf-9667-edf22248b784","added_by":"auto","created_at":"2025-09-01 16:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11282226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/3df0c5bf-1a22-46c1-8641-cacebd0fbcf3.pdf"},{"id":82785117,"identity":"30b15c79-338c-4417-9d8b-f5487ea6c89d","added_by":"auto","created_at":"2025-05-15 09:05:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1\u003c/strong\u003e. (Additional file 1.xlsx) List of genes upregulatedand downregulated in elk calves 2 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/130003b3bd4057c5fe4d148e.xlsx"},{"id":82784769,"identity":"d1ba5006-0e7f-4321-a841-7da773359124","added_by":"auto","created_at":"2025-05-15 08:57:46","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2\u003c/strong\u003e. (Additional file 2.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 2 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/7568111679bb2d50ce4a89ee.xlsx"},{"id":82784774,"identity":"4a39edac-5432-4261-81a1-4083d3903c5f","added_by":"auto","created_at":"2025-05-15 08:57:46","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3\u003c/strong\u003e. (Additional file 3.xlsx) List of genes upregulatedand downregulated in elk calves onday 5 after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/b18b6d3acceb3cabfd9b7784.xlsx"},{"id":82786208,"identity":"436ea439-ae82-4b24-9104-401816693195","added_by":"auto","created_at":"2025-05-15 09:13:47","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":37752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4\u003c/strong\u003e. (Additional file 4.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 5 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/963b50143e39be14da38b7b0.xlsx"},{"id":82784778,"identity":"790458f0-3f72-4a96-8cde-43aa16d527d9","added_by":"auto","created_at":"2025-05-15 08:57:46","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":297097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5\u003c/strong\u003e. (Additional file 5.xlsx) List of genes upregulated and downregulated in elk calves on day 14 after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/138527d49d905213ccadec5f.xlsx"},{"id":82784783,"identity":"d1d66724-7c49-4571-8dee-19462a6f328f","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":37766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 6\u003c/strong\u003e. (Additional file 6.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in elk calves 14 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/c15e0fcee9ddf60c7aa1999f.xlsx"},{"id":82785129,"identity":"f22b9a88-cc30-48fa-b1b3-972f480f6fbf","added_by":"auto","created_at":"2025-05-15 09:05:47","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":112150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7\u003c/strong\u003e. (Additional file 7.xlsx) List of genes upregulatedand downregulated in adult elk on day 2 after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/f26462ed2ec4ec34f779fdf3.xlsx"},{"id":82784781,"identity":"8b8466a0-7dd5-447b-886e-1707a06203f4","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":76997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 8\u003c/strong\u003e. (Additional file 8.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 2 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/33ccd7a25bdf9b3765da38a7.xlsx"},{"id":82785124,"identity":"c44d2d92-4823-4021-974e-a65d3d805dd8","added_by":"auto","created_at":"2025-05-15 09:05:47","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":436707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 9\u003c/strong\u003e. (Additional file 9.xlsx) List of genes upregulatedand downregulated in adult elk on day 5 after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile9.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/bf07545a2811405d6b1d6894.xlsx"},{"id":82784787,"identity":"7405f62a-6e3e-4df6-bc05-3d691c0fdcc4","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":145075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 10\u003c/strong\u003e. (Additional file 10.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 5 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile10.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/c9ab1f3b5907b7cd58bc9349.xlsx"},{"id":82784793,"identity":"5c6101ec-4eee-4e35-969e-874f0a8fc95d","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":455101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 11\u003c/strong\u003e. (Additional file 11.xlsx) List of genes upregulated and downregulated in adult elk on day 14 after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile11.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/6dc1ba1fa066ba9923cee1f2.xlsx"},{"id":82784804,"identity":"9f424bcd-ea6c-4d39-8b36-233835a6918d","added_by":"auto","created_at":"2025-05-15 08:57:47","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":168349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 12\u003c/strong\u003e. (Additional file 12.xlsx) List of GO terms and KEGG pathways associated with up- and downregulated genes in adult elk 14 days after SARS-CoV-2 challenge.\u003c/p\u003e","description":"","filename":"Additionalfile12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6505643/v1/cab2751b8eec89a4814d7960.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Peripheral transcriptional responses to experimental SARS-CoV-2 inoculation in North American Elk cows and calves","fulltext":[{"header":"Background","content":"\u003cp\u003eSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), remains a public health concern and one of the most severe global public health emergencies in history, killing more than 7\u0026nbsp;million people around the world (World Health Organization - \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://data.who.int/dashboards/covid19/deaths\u003c/span\u003e\u003cspan address=\"https://data.who.int/dashboards/covid19/deaths\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by March 2025.\u003c/p\u003e \u003cp\u003eIn addition to its impact on human health, SARS-CoV-2 has also been a concern in various domestic, peridomestic, and wild animals, including cats, dogs, mice, rats, American mink (\u003cem\u003eNeovison vison\u003c/em\u003e), Eurasian river otters (\u003cem\u003eLutra lutra\u003c/em\u003e), ferrets (\u003cem\u003eMustela furo\u003c/em\u003e), Syrian hamsters (\u003cem\u003eMesocricetus auratus\u003c/em\u003e), gorillas, lions, tigers, Virginia opossums (\u003cem\u003eDidelphis virginiana\u003c/em\u003e), raccoons (\u003cem\u003eProcyon lotor\u003c/em\u003e), groundhog (\u003cem\u003eMarmota monax\u003c/em\u003e), red bats (\u003cem\u003eLasiurus borealis\u003c/em\u003e), white-tailed deer (\u003cem\u003eOdocoileus virginianus\u003c/em\u003e), red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e), and fallow deer (\u003cem\u003eDama dama\u003c/em\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At the molecular level, the most defining structural protein of SARS-CoV-2 is the spike (S) protein. During infection, the S protein binds to surface angiotensin-converting enzyme 2 (ACE2), the receptor responsible for virus entrance into the cell, facilitating virus replication [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The variability in the specificity of the interaction between viruses and receptors is believed to reflect the range of susceptible hosts. The ACE2 protein of humans has a high degree of homology with that of white-tailed deer, reindeer, and Pierre Davids\u0026rsquo;s deer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. White-tailed deer were previously shown to be highly susceptible to infection with viral shedding and transmission to other white-tailed deer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Subsequent surveys of wild white-tailed deer across the United States (US) revealed widespread exposure/infection, which suggests ongoing deer-to-deer transmission and a possible source of deer-to-human infection.\u003c/p\u003e \u003cp\u003eAlthough white-tailed deer are the most abundant wild ungulate in North America, with approximately 30\u0026nbsp;million found in the US alone, another closely related but less numerous large ungulate is North American elk (\u003cem\u003eCervus elaphus canadensis\u003c/em\u003e). In a recent publication [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] from our own laboratory, the susceptibility of calves and adult elk to SARS-CoV-2 infection was investigated. Although elk are moderately permissive to SARS-CoV-2 infection and exhibit seroconversion, viral shedding and tissue distribution are much lower than those observed in white-tailed deer. Interestingly, viral RNA and viral protein were detectable in lymphoid tissues 21 days after infection in both elk calves and adults. Based on these findings and to further understand virus‒host interactions, we investigated the changes in whole-blood gene expression in elk calves and adults following experimental challenge with SARS-CoV-2. Understanding the transcriptomic response to infection could shed light on SARS-CoV-2 pathophysiology in mammals, including age-related differences, which have not been investigated previously.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe evaluated the RNA-seq whole-blood transcriptomes of calves and adult elk cows experimentally infected with USA-WA1/2020 SARS-CoV-2, on days 0, 2, 5, and 14 post inoculation (pi).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferentially expressed genes between day 0 and days 2, 5, and 14 post-SARS-CoV-2 inoculation in elk calves\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn elk calves, the comparison of day 2 pi to day 0 revealed 715 genes that were significantly differentially expressed (DE), with 183 downregulated genes and 532 upregulated genes (p-adjusted\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, see Additional file 1). Among the upregulated genes, 24 were involved in the coronavirus disease pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway) (Additional file 2). Interestingly, upregulated genes in this pathway included regulators of a \u0026ldquo;cytokine storm\u0026rdquo; immune response to SARS-CoV-2, such as \u003cem\u003eCXCL10\u003c/em\u003e (log2fc\u0026thinsp;=\u0026thinsp;2.60) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eIRF3\u003c/em\u003e (interferon regulatory factor 3, log2fc\u0026thinsp;=\u0026thinsp;0.36), \u003cem\u003eIRF9\u003c/em\u003e (interferon regulatory factor 9, log2fc\u0026thinsp;=\u0026thinsp;1.00), \u003cem\u003eMX1\u003c/em\u003e (MX Dynamin Like GTPase 1, log2fc\u0026thinsp;=\u0026thinsp;3.70) and \u003cem\u003eMX2\u003c/em\u003e (MX Dynamin Like GTPase 2, log2fc\u0026thinsp;=\u0026thinsp;4.70). Both \u003cem\u003eMX1\u003c/em\u003e and \u003cem\u003eMX2\u003c/em\u003e are linked to the cellular antiviral immune response [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, \u003cem\u003eISG15\u003c/em\u003e (ISG15 ubiquitin-like modifier, log2fc\u0026thinsp;=\u0026thinsp;3.90) was upregulated and has previously been implicated in inflammatory responses after SARS-CoV-2 exposure [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with day 0, the day 5 pi analysis revealed 921 DE genes; 339 were downregulated, and 582 were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, see Additional file 3). Several upregulated genes were associated with immune response pathways associated with viral responses, including the NOD-like receptor signaling pathway (20 genes), herpes simplex virus 1 infection (20 genes), Epstein‒Barr virus infection (19 genes), hepatitis C (17 genes), influenza A (17 genes), human papillomavirus infection (16 genes), and the biological term for defense response to virus (12 genes) (see Additional file 4).\u003c/p\u003e \u003cp\u003eA comparison of day 14 pi versus day 0 revealed the greatest number of DEGs, with a total of 7,179 DEG, of which 3,602 were downregulated and 3,577 upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, see Additional file 5). Some of the upregulated genes were associated with known SARS-CoV-2 players, such as \u003cem\u003eIRAK1\u003c/em\u003e (interleukin 1 receptor-associated kinase 1, log2fc\u0026thinsp;=\u0026thinsp;0.87), \u003cem\u003eIRF3\u003c/em\u003e (interferon regulatory factor 3, log2fc\u0026thinsp;=\u0026thinsp;0.59), \u003cem\u003eISG15\u003c/em\u003e (ISG15 ubiquitin-like modifier, log2fc\u0026thinsp;=\u0026thinsp;1.30) and \u003cem\u003eIRF9\u003c/em\u003e (interferon regulatory factor 9, log2fc\u0026thinsp;=\u0026thinsp;0.66). Additionally, upregulated genes were associated with protein binding, ATP binding, structural constituents of ribosomes, and translation GO terms, as well as thermogenesis and ribosomes as KEGG pathways, all of which are associated with viral responses. Interestingly, several upregulated DEGs were related to neurodegenerative diseases, such as genes related to Alzheimer\u0026rsquo;s disease: \u003cem\u003ePSEN2\u003c/em\u003e, \u003cem\u003eTREM2\u003c/em\u003e, and \u003cem\u003eGAB2\u003c/em\u003e [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; Parkinson\u0026rsquo;s disease: \u003cem\u003eATP13A2\u003c/em\u003e and \u003cem\u003eDCTN1\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; and amyotrophic lateral sclerosis: \u003cem\u003eTAF15\u003c/em\u003e and \u003cem\u003eFUS\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] (see Additional file 6).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferentially expressed genes between day 0 and days 2, 5, and 14 post SARS-CoV-2 inoculation in adult elk\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCompared with those of day 0 samples, the DEG analysis of day 2 pi samples revealed 2,756 DE genes (see Additional file 7). In total, 1,421 genes were upregulated, whereas 1,234 genes were downregulated (p adjusted\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The upregulated genes were associated with coronavirus disease KEGG pathways, including members of the MAPK family of cascades, such as \u003cem\u003eMAPK3\u003c/em\u003e (mitogen-activated protein kinase 3) and \u003cem\u003eMAPKAPK3\u003c/em\u003e (MAPK activated protein kinase 3) (with log2-fold change values\u0026thinsp;=\u0026thinsp;0.70 and 0.47, respectively); \u003cem\u003eMyd88\u003c/em\u003e (myeloid differentiation primary response gene, log2fc\u0026thinsp;=\u0026thinsp;0.57); \u003cem\u003eIRF3\u003c/em\u003e (interferon regulatory factor 3, log2fc\u0026thinsp;=\u0026thinsp;0.84); \u003cem\u003eISG15\u003c/em\u003e (ISG15 ubiquitin-like modifier, log2fc\u0026thinsp;=\u0026thinsp;4.94); \u003cem\u003eSTAT1\u003c/em\u003e (signal transducer and activator of transcription 1, log2fc\u0026thinsp;=\u0026thinsp;0.99); and \u003cem\u003eSTAT2\u003c/em\u003e (signal transducer and activator of transcription 2, log2fc\u0026thinsp;=\u0026thinsp;1.00). Most of these genes are involved in various immune system pathways, including antiviral defense (see Additional file 8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis revealed 9,708 DE genes in the adult elk between day 5 pi and day 0; of these genes, 5,399 were downregulated, and 4,309 genes were upregulated (p-adjusted\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, see Additional file 9). Several genes presented very large log2-fold changes in both directions (up- and downregulated). Examples include \u003cem\u003eVIM\u003c/em\u003e (vimentin, log2fc = -17.77), \u003cem\u003eADA\u003c/em\u003e (adenosine deaminase, log2fc = -16.47), \u003cem\u003eTRIM15\u003c/em\u003e (tripartite motif containing 15, log2fc\u0026thinsp;=\u0026thinsp;17.82), \u003cem\u003eFAM227A\u003c/em\u003e (family with sequence similarity 227 Member A, log2fc\u0026thinsp;=\u0026thinsp;15.70) and \u003cem\u003eMAJIN\u003c/em\u003e (membrane anchored junction protein, log2fc\u0026thinsp;=\u0026thinsp;15.29). Extended results can be found in Additional file 10. Among the genes upregulated for this comparison, 106 were associated with the coronavirus disease KEGG pathway and included \u003cem\u003eACE2\u003c/em\u003e (angiotensin-converting enzyme 2, log2fc\u0026thinsp;=\u0026thinsp;2.87), \u003cem\u003eTNF\u003c/em\u003e (tumor necrosis factor, log2fc\u0026thinsp;=\u0026thinsp;3.46), and MAPK family members, such as \u003cem\u003eMAPK4\u003c/em\u003e (mitogen-activated protein kinase 4, log2fc\u0026thinsp;=\u0026thinsp;9.34), \u003cem\u003eMAPK11\u003c/em\u003e (mitogen-activated protein kinase 11, log2fc\u0026thinsp;=\u0026thinsp;10.06), and \u003cem\u003eISG15\u003c/em\u003e (ISG15 ubiquitin-like modifier, log2fc\u0026thinsp;=\u0026thinsp;3.21).\u003c/p\u003e \u003cp\u003eBetween day 14 pi and day 0, 3,950 genes were DE; 1,799 were downregulated, and 2,151 were upregulated, with a p-adjusted\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, see Additional file 11). As observed on day 5 pi, there were also large log2-fold changes in \u003cem\u003eALAS2\u003c/em\u003e (5'-Aminolevulinate Synthase 2, log2fc = -19.11), \u003cem\u003eFTH1\u003c/em\u003e (Ferritin Heavy Chain 1, log2fc = -19.03), \u003cem\u003eGNAI2\u003c/em\u003e (G Protein Subunit Alpha I2, log2fc = -18.78), \u003cem\u003eUCP3\u003c/em\u003e (Uncoupling Protein 3, log2fc\u0026thinsp;=\u0026thinsp;16.43), \u003cem\u003eMAJIN\u003c/em\u003e (Membrane Anchored Junction Protein, log2fc\u0026thinsp;=\u0026thinsp;15.04) and \u003cem\u003eH1-1\u003c/em\u003e (H1.1 Linker Histone, Cluster Member, log2fc\u0026thinsp;=\u0026thinsp;14.69). KEGG pathway analysis revealed 101 upregulated DEGs associated with the coronavirus disease - COVID-19 KEGG pathway, including \u003cem\u003eMAS1\u003c/em\u003e (MAS1 Proto-Oncogene, G Protein-Coupled Receptor, log2fc\u0026thinsp;=\u0026thinsp;10.46), \u003cem\u003eTNF\u003c/em\u003e (Tumor necrosis factor, log2fc\u0026thinsp;=\u0026thinsp;4.82), MAPK family members such as \u003cem\u003eMAPK11\u003c/em\u003e (Mitogen-Activated Protein Kinase 11, log2fc\u0026thinsp;=\u0026thinsp;7.10) and \u003cem\u003eMAPK12\u003c/em\u003e (Mitogen-Activated Protein Kinase 12, log2fc\u0026thinsp;=\u0026thinsp;4.69). Genes such as \u003cem\u003eUCP3\u003c/em\u003e (uncoupling protein 3), \u003cem\u003eMAJIN\u003c/em\u003e (membrane anchored junction protein), \u003cem\u003eH1-1\u003c/em\u003e (H1.1 linker histone, cluster member) and \u003cem\u003eHAS1\u003c/em\u003e (hyaluronan synthase 1) presented log2fc values of 16.43, 15.04, 14.69 and 14.32, respectively. \u003cem\u003eISG15\u003c/em\u003e was also upregulated, with log2fc\u0026thinsp;=\u0026thinsp;1.17 (see Additional file 12).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eComparison of differentially expressed genes between elk calves and adults\u003c/h2\u003e \u003cp\u003eWe also compared the overlap between significantly up- and down-regulated DE genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), within timepoints between adults and calves to understand the similarities and differences in the immune response. When the downregulated genes between the adult and calf groups on day 2 pi vs. day 0 were compared, 35 genes overlapped. Most of them associated with the Golgi apparatus, including \u003cem\u003eSTK26\u003c/em\u003e (serine/threonine-protein kinase 26) and \u003cem\u003eHOOK3\u003c/em\u003e (hook microtubule tethering protein 3) but also include \u003cem\u003eGEMIN6\u003c/em\u003e (gem nuclear organelle-associated protein 6), a gene already associated with SARS-CoV-2 infection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Among the 239 upregulated genes shared between adults and calves, 19 genes were related with the coronavirus disease-COVID-19 KEGG pathway, and the others were associated with additional antiviral responses, including herpes simplex virus 1 infection, influenza A, the defense response to a virus, hepatitis C, and NOD-like receptor signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen DE gene expression from both ages of elk was compared between day 5 pi and day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), 170 downregulated genes were shared. Many of these genes had GO terms related to the nucleus, regulation of transcription by RNA polymerase II, DNA-binding transcription factor activity, RNA polymerase II-specific and ATP binding. The associated KEGG pathways included the regulation of the actin cytoskeleton, cellular senescence and the MAPK signaling pathway. On the upregulated side, 164 genes were shared between adults and calves, 10 of which were associated with the KEGG pathways for coronavirus disease-2019 (COVID-19), other genes related to the NOD-like receptor signaling pathway, and different viral infections, such as influenza A, hepatitis C, Epstein\u0026ndash;Barr virus infection, and herpes simplex virus 1 infection.\u003c/p\u003e \u003cp\u003eA comparison of the adult and calf DE gene lists between day 14 pi and day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) revealed that 2,241 downregulated genes were shared. The genes with reduced expression were associated with KEGG pathways related to viral infections, including herpes simplex virus 1 disease, human papillomavirus infection, and human cytomegalovirus infection, and with GO terms associated with the nucleus, protein binding, cytoplasm, ATP binding and regulation of transcription by RNA polymerase II. Among the 1,043 shared upregulated DE genes, more than 100 genes were associated with metabolic KEGG pathways, and many of them had GO terms related to the cytoplasm, cytosol and mitochondrion. Additionally, 34 genes were involved in pathways associated with neurodegeneration, including multiple diseases, such as 29 genes associated with Alzheimer\u0026rsquo;s disease, 28 genes associated with Huntington\u0026rsquo;s disease, and 26 genes associated with prion disease and Parkinson\u0026rsquo;s disease.\u003c/p\u003e \u003cp\u003eFinally, we determined which DE genes presented up- and downregulated expression levels across both groups and all timepoints. In total, 7 genes were found to be upregulated in both groups across all time points: \u003cem\u003eG6PC3, IFI35, LOC122430223, IRF9, LGALS9, DHX58 and ISG15\u003c/em\u003e. In the downregulated group, 6 DE genes overlapped between adults and calves at different timepoints: \u003cem\u003eSTK26\u003c/em\u003e, \u003cem\u003eEDEM3\u003c/em\u003e, \u003cem\u003eADSS2\u003c/em\u003e, \u003cem\u003eCPNE3, STRN and ADAM10\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cem\u003eIngenuity Pathway Analysis of differentially expressed genes between adult elk and calves\u003c/em\u003e\u003c/div\u003e \u003cp\u003eTo further analyze the elk response to SARS-CoV-2, we used the Ingenuity Pathway Analysis (IPA) software to examine changes in the coronavirus pathogenesis pathway. We compared the predicted intracellular responses at different stages of infection over time and between calves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and adult elk (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn calves, DE genes between day 2 pi and day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) suggest activation of the innate immune system. The molecular activity predictor tool highlighted the upregulation of the signal transducers and activators of transcription (STAT) pathway, with the \u003cem\u003eSTAT1\u003c/em\u003e and \u003cem\u003eSTAT2\u003c/em\u003e genes activated, which are known mediators of antiviral host defense and interferon response signaling. Similarly, the terms \u003cem\u003eIFN-1\u003c/em\u003e and \u003cem\u003eISGF3\u003c/em\u003e were activated, contributing to proinflammatory NF-kB and interferon type 1 responses. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows the comparison of DE genes from day 5 pi and day 0, where the \u003cem\u003eISGF3\u003c/em\u003e cascade remained activated in calves, along with \u003cem\u003eSTAT1\u003c/em\u003e, \u003cem\u003eSTAT2, NF-kB\u003c/em\u003e and Interferon type 1 responses. The IPA predicted the inhibition of SARS-CoV-2 replication and continued activation of the innate and adaptive immune responses, including associations with pathway terms such as apoptosis and cytokine storms by hypercytokinaemia. A comparison of day 14 versus day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) revealed broad activation of pathway terms related to infection: \u003cem\u003eIFN-1\u003c/em\u003e remained active, and SARS-CoV-2 replication was still predicted to be inhibited. Transforming growth factor (TGF)-beta receptor activity was inhibited, which was linked to the downregulation of the \u003cem\u003eSMAD3\u003c/em\u003e gene. Notably, in calves, the spike protein receptor ACE2, which is necessary for virus entry into the cell, was predicted to be activated only on day 14 DE gene list.\u003c/p\u003e \u003cp\u003eIn adult elk, inflammatory signals associated with infection were more broadly activated in the IPA analysis of DE genes between day 2 pi and day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The \u003cem\u003eTGFB\u003c/em\u003e, \u003cem\u003eAGTR4 STAT1\u003c/em\u003e, \u003cem\u003eSTAT2\u003c/em\u003e, and \u003cem\u003eISGF3\u003c/em\u003e genes were upregulated, along with Interferon type 1, adaptive, and innate immune activation. Interestingly, in contrast to that in calves, the \u003cem\u003eACE2\u003c/em\u003e receptor gene was predicted to be activated earlier in adult animals as compared to the calves. A comparison of the DE gene predictions for day 5 pi and day 0 revealed a marked decrease in the immune response in adult animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). A \u0026lsquo;deactivation\u0026rsquo; of Interferon type 1 response was seen, as well as a decrease in the involvement of \u003cem\u003eSTAT1\u003c/em\u003e, \u003cem\u003eSTAT2, ISGF3\u003c/em\u003e, \u003cem\u003eTGFB\u003c/em\u003e and the \u003cem\u003eAGTR4\u003c/em\u003e receptor. By day 5 pi in adults, the \u003cem\u003eACE2\u003c/em\u003e receptor and interferon type 1 response were still activated. However, in the DE gene lists from day 14 pi, the \u003cem\u003eACE2\u003c/em\u003e receptor gene was no longer activated, and the interferon type 1 response was inhibited along with a decrease in the expression level of other inflammatory genes, such as NF\u003cem\u003e-kB\u003c/em\u003e, \u003cem\u003eSTAT1\u003c/em\u003e and \u003cem\u003eISGF3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we characterize for the first time the changes in the peripheral blood gene expression profile of calves and adult elk experimentally challenged with SARS-CoV-2. Our data illustrates a broad and rapid immune response to SARS-CoV-2 inoculation. Gene expression profiles highlight the changing expression of genes related to important pathways for controlling viral infection and activating the host immune system response. These data offer an important resource for comparative studies in elk and other mammals, including humans, to help understand the molecular machinery behind the antiviral response.\u003c/p\u003e \u003cp\u003eThe differentially expressed gene analysis consistently revealed upregulated genes involved in SARS-CoV-2 infection processes and Coronavirus disease KEGG pathways in both calves and adult elk. In a recent publication from our group [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], both elk calves and adults are susceptible to infection and have the ability to develop neutralizing antibodies against an ancestral SARS-CoV-2 variant (USA-WA1/2020). Interestingly, however, calves had a higher neutralizing response compared to the adults. Additionally, viral RNA and viral protein persisted in the retropharyngeal lymph nodes of both adults and calves.\u003c/p\u003e \u003cp\u003eOur results suggest that age may have a significant impact on the elk response to SARS-CoV-2 challenge. The literature defines significant age-dependent differences in the immune response reported in humans [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], where older individuals present more evidence of high morbidity and mortality than do younger individuals, who have a more effective immune response to spike and peptides against SARS-CoV-2. The severity of coronavirus disease (COVID-19) in adult humans could result from the decreased potential to immunomodulate excessive inflammation after strong cytokine secretion by T cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consistent with those findings, in our study, the variation in the transcriptomic response between calves and adult elk was illustrated by the substantial increase in DE genes at all timepoints in the adult elk and the lack of conservation across DEGs from both ages. In the elk calves, a relatively small number of genes were DE; however, immune activation and response trends were evident. Within the day 2 pi comparison, the upregulated genes were associated with GO terms and KEGG pathways related to coronavirus disease, the defense response to virus, and influenza A and herpes simplex virus 1 infection. Viral activation was more emphasized for calves on day 5 pi, when there was a significant increase in the expression of genes related to an antiviral immune response, such as genes from the interferon regulatory factor (IFR) family, interferon gamma-inducible proteins and other interferon-stimulated genes (ISGs). Notably, in calves on day 14 pi, there was an increased number of DE upregulated genes associated with neurodegenerative disease KEGG pathways. While we did not observe any clinical signs associated with neurological disease in the calves, it is worth noting that in humans, neurological symptoms and neuroinflammatory events have been described during and following infection with SARS-CoV-2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn adult elk, many DE genes were related to coronavirus disease - COVID-19 KEGG pathway and participating in immune system activation and the defense response to this coronavirus and other viral diseases. The GO and KEGG terms associated with COVID-19 appeared as early as day 2 pi. By day 5 pi, we observed larger log2-fold changes in expression, and genes related again to coronavirus disease \u0026ndash; COVID-19 KEGG pathway. Some of the genes with larger log2-fold change are important to the viral and bacterial pathogens infections. \u003cem\u003eVIM\u003c/em\u003e (Vimentin) have been shown to facilitate the binding of pathogens to the cell surface and promote their persistence in the host cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and \u003cem\u003eTRIM15\u003c/em\u003e is a member of the TRIM family, a new class of antiviral proteins and essential components for innate antiviral immunity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The impact of infection on day 14 pi is still evident, with log2-fold changes of approximately\u0026thinsp;+\u0026thinsp;16 for upregulated genes and \u0026minus;\u0026thinsp;19 for downregulated genes, which were still associated with coronavirus disease pathways. This further highlight possible differences in immune responses, including the longevity of infection, between young and adult individuals across species.\u003c/p\u003e \u003cp\u003eSignificantly DE genes conserved between calves and adults could be potential biomarkers of SARS-CoV-2 infection in wildlife and/or other hosts. Across calves and adult elk, seven upregulated DEGs were shared across all time points: \u003cem\u003eG6PC3, IFI35, LOC122430223, IRF9, LGALS9, DHX58 and ISG15\u003c/em\u003e. Glucose-6-phosphatase catalytic subunit 3 \u003cem\u003e(G6PC3)\u003c/em\u003e is associated with macrophage activation, regulation of signal transduction, positive regulation of the defense immune response, and innate immune system activation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Interferon-induced protein 35 (\u003cem\u003eIFI35\u003c/em\u003e) is involved in the type I interferon signaling pathway and has been shown to be upregulated in patients with mild clinical COVID-19 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Interferon regulatory factor 9 (\u003cem\u003eIRF9\u003c/em\u003e) is an important component of the type I and III interferon signaling pathways, and it has been associated with \u003cem\u003eSTAT1\u003c/em\u003e and STAT2\u0026mdash;also significantly DE genes in our study\u0026mdash;to form the trimeric transcription factor \u003cem\u003eISGF-3\u003c/em\u003e, which is associated with cellular antiviral responses [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The \u003cem\u003egalectin-9\u003c/em\u003e gene (\u003cem\u003eLGALS9\u003c/em\u003e) is a beta-galactoside-binding protein that is involved in cell signaling, adhesion, and migration and is involved in inflammatory processes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The LGALS9 protein has already been described as a potential biomarker for many diseases, including COVID-19, and is highly expressed in hospitalized patients [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Another study revealed that this gene potentially enhances virus replication and inflammation in epithelial cells \u003cem\u003ein vitro\u003c/em\u003e, suggesting that galectin-9 impacts the early stage of SARS-CoV-2 infection [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The DExH-box helicase 58 (\u003cem\u003eDHX58\u003c/em\u003e) gene is a member of a large family of RNA helicases and is responsible for encoding the LGP2 protein, which is important for RNA metabolism, splicing, recognition, modification, degradation, transport, and translation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. LGP2 proteins are involved in immunity against viruses. A study using a transgenic mouse model overexpressing this protein revealed a significant reduction in inflammatory mediators and low leucocyte infiltration into the bronchoalveolar space, resulting in a significant survival advantage compared with the wild-type group and leading to a reduction in the expression of the influenza A virus inflammatory response [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Like influenza A virus, SARS-CoV-2 results in infection of respiratory epithelial cells, so increased production of \u003cem\u003eLGP2\u003c/em\u003e genes could help reduce the incidence of clinical disease. The \u003cem\u003eISG15\u003c/em\u003e gene was upregulated at all post challenge timepoints, with high log2-fold changes early after inoculation (\u003cem\u003ei.e.\u003c/em\u003e, 3.90 at 2 pi in calves and 4.94 at 2 pi in adults). As an IFN-stimulated gene (ISG), \u003cem\u003eISG15\u003c/em\u003e is a key orchestrator of immune defense during viral infection and has already been described participating in the SARS-CoV-2 infection, inflammation, and immune responses [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eISG15\u003c/em\u003e plays a vital role in cell cycle regulation, maintenance of genome stability, and innate immune signaling after viral infection [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. An additional conserved gene that was DE in both calves and adults across all time points and has previously been reported in relation to SARS-CoV-2 infection was \u003cem\u003eTNFAIP3\u003c/em\u003e (tumor necrosis factor, alpha-induced protein 3). Sarlo-Davila et al. observed that the \u003cem\u003eTNFAIP3\u003c/em\u003e gene was upregulated in deer respiratory epithelial cells \u003cem\u003ein vitro.\u003c/em\u003e In our study, \u003cem\u003eTNFAIP3\u003c/em\u003e was downregulated on day 5 (log2fc=-6.06) and day 14 (log2fc=-14.08) pi in adult elk, whereas the same gene was upregulated (log2fc\u0026thinsp;=\u0026thinsp;0.40) on day 2 and downregulated (log2fc=-0.44) on day 14 pi in the calf group.\u003c/p\u003e \u003cp\u003eIt is known that some viruses can downregulate components of the immune system, including the receptor necessary for host cell entry, to prevent reinfection of the same cell [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Here, we also investigated the downregulated DE genes shared between calves and adult elk at different timepoints and identified six genes: \u003cem\u003eSTK26\u003c/em\u003e, \u003cem\u003eEDEM3\u003c/em\u003e, \u003cem\u003eADSS2\u003c/em\u003e, \u003cem\u003eADAM10\u003c/em\u003e, \u003cem\u003eCPNE3\u003c/em\u003e and \u003cem\u003eSTRN\u003c/em\u003e. The \u003cem\u003eSTK26\u003c/em\u003e (Serine/Threonine Kinase 26), also known as MST4, is a protein kinase that is expressed in monocytes and T, B and NK cells and mediates bacterial infections [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Compared with that in asymptomatic patients, the expression of \u003cem\u003eSTK26\u003c/em\u003e is increased in sick human patients after SARS-CoV-2 infection [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A study investigating drug target proteins revealed that SARS-CoV-2 infection can change the structure of endoplasmic reticulum (ER) proteins at the initiation of virus‒cell interactions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. \u003cem\u003eEDEM3\u003c/em\u003e (ER Degradation Enhancing Alpha-mannosidase Like Protein 3) is a member of a group of proteins that perform glycoprotein degradation in the ER [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]; \u003cem\u003eEDEM3\u003c/em\u003e was downregulated in our study, indicating the possible interference of the virus in protein production. Adenylosuccinate synthase (ADSS) is known as a convertor of inosine monophosphate to adenosine monophosphate (AMP) and is present in vertebrates as two different isozymes: \u003cem\u003eADSS1\u003c/em\u003e, which is the basic form that participates in the purine nucleotide cycle, and the acidic form \u003cem\u003eADSS2\u003c/em\u003e, which catalyzes the synthesis of AMP [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Another downregulated gene shared between the groups is \u003cem\u003eCopine 3\u003c/em\u003e (\u003cem\u003eCPNE3\u003c/em\u003e), a member of the CNPE family; this gene is strongly expressed in the early stage of neutrophil differentiation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and plays an important role in maintaining the normal function of pancreatic β-cells, regulating glucose uptake and insulin secretion [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eADAM10\u003c/em\u003e (ADAM metallopeptidase domain 10) is an adhesion receptor, and the mRNA that encodes this gene has been found in immune cells and is involved in the activation, differentiation and migration of T cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. ADAM proteins, important membrane-anchored enzymes that play important roles in converting cytokine precursors near the membrane into soluble bioactive proteins, mediate the regulation of the post-translational function of proteins in the immune system [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The \u003cem\u003eADAM10\u003c/em\u003e gene is also involved in the regulation of cytokine production and alternative protease cleavage, characterizing some important host dependency factors for SARS-CoV-2 entry into cells [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIngenuity pathway analysis illustrated the participation of DE genes in the activation of adaptive and innate immune responses during SARS-CoV-2 infection. As soon as day 2 pi, both groups of elk had signatures consistent with innate and adaptive immune activation as well as the inhibition of the replication of SARS-CoV-2, which is consistent with most host approaches to control infection. Specifically, interferon-associated terms such as Interferon Stimulated Factor 3 (\u003cem\u003eISGF3)\u003c/em\u003e, NF-kB, and general interferon type 1 responses were consistently activated. Type I interferons and associated mediators are known to play protective roles in controlling viral infections [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. One of those genes is \u003cem\u003eIRF3\u003c/em\u003e (interferon regulatory factor 3), the primary early regulatory factor responsible for inducing IFN-1 during viral infection, which positively regulates apoptosis and inhibits NF-kB translocation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cem\u003eIRF7\u003c/em\u003e (interferon regulatory factor 7) can mediate inflammatory responses, and a lack of \u003cem\u003eIRF9\u003c/em\u003e results in increased COVID-19 risk [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Broadly, the host immune response, inflammatory, and antiviral signaling pathways were the highest early in the adult elk when the day 2 pi profile was examined, but they appeared to decline over time, as evidenced by the inhibition of immune terms by day 5 pi. In contrast, the calves appeared to show an increase in the immune response and interferon activation through day 5 pi and only moderately decreased in activity by day 14 di. Notably, adults showed a predicted involvement of \u003cem\u003eACE2\u003c/em\u003e as early as day 2 and through day 5 pi, whereas calves showed activation of \u003cem\u003eACE2\u003c/em\u003e only on day 14 pi.\u003c/p\u003e \u003cp\u003eBoth calves and adult elk have been shown to lack clinical signs associated with SARS-CoV-2 disease, suggesting that while elk can become infected, it is successful at controlling the infection without causing disease. Collectively, this work provides unique insight into potential mechanisms of viral control by the host immune system, variation in disease responses among age groups, and gene signature associations with stages of infection.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHere, for the first time, we describe the peripheral transcriptional response of North American elk to SARS-CoV-2 infection at different timepoints and in different age groups. Overall, this work facilitates the discussion of species-specific responses to a zoonotic pathogen with global human health implications, provides further characterization of wildlife immune responses, and highlights the gene expression profiles associated with SARS-CoV-2.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElk challenge and sample collection\u003c/h2\u003e \u003cp\u003e All procedures and animal work were performed following approval by the NADC Animal and Care Use Committee (IACUC) and follow the Guide for Care and Use of Laboratory Animals regulations. For the challenge studies, adult elk (n\u0026thinsp;=\u0026thinsp;10), approximately 4 years of age, were transferred to agricultural biosafety level 3 (AgBSL3) facilities and allowed to acclimate for 2 weeks. Elk calves (n\u0026thinsp;=\u0026thinsp;11), approximately 5 months of age, were born in high containment, and remained with dams until weaning. All animals were challenged with an ancestral strain of SARS-CoV-2 (USA-WA1/2020), as described previously in Boggiatto et al. (2024). Briefly, the animals were challenged intranasally with a SARS-CoV-2 isolate (USA-WA1/2020 \u0026ndash; SARS-Related Coronavirus 2, Isolates hCoV-19/USA-WA1/2020). Blood samples were collected from elk calves and elk adult cows pre-challenge day 0 (control), day 2, day 5, and day 14 post- inoculation using PAXgene\u0026reg; Blood RNA tubes (BD Biosciences, San Jose, CA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA isolation, library preparation and sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using MagMAX\u0026trade; for Stabilized Blood Tubes RNA Isolation Kit, which is compatible with PAXgene\u0026trade; Blood RNA Tubes (catalog number 4451894) (Thermo Fisher Scientific, Waltham, MA). The RNA integrity was determined via a bioanalyzer, and the average RNA integrity number (RIN) for all the samples was 9.3. Library preprocessing was performed via an Illumina mRNA kit, and sequencing was performed at the University of Illinois, Urbana-Champaign, at Roy J. Carver Biotechnology Center via Nova Seq X Plus, which generated 150 base pair paired-end reads.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing bioinformatics: quality control and read mapping\u003c/h3\u003e\n\u003cp\u003eData processing was performed using the Nextflow (version 24.04.2) nf-core/rna-seq pipeline (version 3.14.0) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Read quality control (QC) was processed by FastQC software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 0.12.1), and the library adaptors were trimmed using TRIMgalore software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 0.6.7), removing the reads with Phred quality scores lower than 20. After filtering, the sequence reads were aligned against the \u003cem\u003eCervus canadensis\u003c/em\u003e genome assembly (ASM19320065v1) via STAR software [53, version 2.7.10a]. Gene counts were performed with featureCounts software [54, release 2.0.4], and all subsequent statistical analyses were performed in the R environment.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferentially expressed gene analysis\u003c/h2\u003e \u003cp\u003eThe differentially expressed (DE) analysis was performed comparing each challenged day post-inoculation (day 2, day 5 and day 14) to the control group (day 0) for both the adult and calf groups, using DESeq2 software in R [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], version 1.44.0. For the DE analysis, reads were filtered to remove genes with no expression (zero reads), genes with very low expression (less than 1 read per sample on average), and genes with rare expression across the samples (genes with read counts that were not present in at least 3 samples).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGO term enrichment analysis and Ingenuity Pathway Analysis\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis and KEGG pathway analysis were performed using DAVID bioinformatics software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/tools.jsp\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/tools.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to help interpret and summarize the biological processes and pathways associated with the DE genes. The overlap between DE genes in the different groups was determined via Venny [56, version 2.1]. Ingenuity pathway analysis (IPA) (QIAGEN, Redwood City, CA, USA) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] was performed using the gene expression data as input to predict the activation or inhibition of the metabolic canonical pathways in a core analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures and animal work were performed following approval by the NADC Animal and Care Use Committee (IACUC) and follow the Guide for Care and Use of Laboratory Animals regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is available in the SRA-NCBI repository under project number PRJNA1196870 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1196870).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by intramural funding from the U.S. Department of Agriculture and American Rescue Plan (ARP) funds through an interagency agreement between the Agricultural Research Service (ARS) and the Animal and Plant Health Inspection Service (APHIS) Wildlife Service (WS). Additionally, this research was supported in part by an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE-SC0014664. All the opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of the USDA, DOE, or ORAU/ORISE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.P. analyzed data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003eK.M.S.D. analyzed data and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eA.C.B. participated in study design, prepared the virus for infections, assisted with animal work, sample collection, and sample processing.\u003c/p\u003e\n\u003cp\u003eE.D.C. participated in study design, collected samples.\u003c/p\u003e\n\u003cp\u003eS.C.O. performed animal experiments and collected samples.\u003c/p\u003e\n\u003cp\u003eP.M.B. participated in study design, performed the animal experiments, collected samples, analyzed data, and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eM.V.P. obtained funding, managed project, participated in study design, performed animal experiments, and collected samples.\u003c/p\u003e\n\u003cp\u003eE.J.P. participated in the study design, processed samples, analyzed data, and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the National Animal Disease Center (NADC) Animal Resources Unit (ARU) for the expert care of the animals used in this study. Specifically, the authors thank Dr. Rebecca Cox, Derek Vermeer, Jonathan Gardner, Tiffany Williams and Kolby Stallman for their animal husbandry, care, and assistance. Additionally, we would like to thank Sue Osorio and Sarah Anderson for their excellent technical assistance. The USDA is an equal opportunity provider and employer. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoldberg AR, Langwig KE, Brown KL, Marano JM, Rai P, King KM, et al. Widespread exposure to SARS-CoV-2 in wildlife communities. Nat Commun. 2024;15.\u003c/li\u003e\n\u003cli\u003eWHOA. SARS-COV-2 IN ANIMALS \u0026ndash; SITUATION REPORT 22 SARS-COV-2 IN ANIMALS \u0026ndash; SITUATION REPORT 22. 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Bioinformatics. 2014;30:523\u0026ndash;30.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, coronavirus, RNA-seq, zoonotic, elk, Cervus elaphus","lastPublishedDoi":"10.21203/rs.3.rs-6505643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6505643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a health risk for humans and other domestic and wildlife species. Recently, North American elk has been identified as seropositive for SARS-CoV-2, thus posing a potential threat to humans and other mammals. In this work, we characterized the peripheral transcriptomic response to experimental SARS-CoV-2 infection in calves and adult elk.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSignificantly differentially expressed genes were identified at 2, 5, and 14 days post inoculation (dpi) for both age groups. Adult elk presented the greatest number of differentially expressed (DE) genes at all time points, including many genes associated with the viral response, immune activation, and antibody production, those associated with coronavirus disease (COVID-19), and coronavirus GO terms and KEGG pathways. Calves presented DE genes associated with viral responses at 5 dpi as well as neurodegenerative-associated genes at 14 dpi. Both adults and calves showed predicted activation of the \u003cem\u003eISGF3\u003c/em\u003e and \u003cem\u003eIFN type I\u003c/em\u003e pathways at 2 dpi and, globally, increased activity related to the coronavirus pathway disease at 5 and 14 dpi.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, this work provides valuable data characterizing the immune response of elk to viral diseases as well as the response of wildlife to SARS-CoV-2 infection.\u003c/p\u003e","manuscriptTitle":"Peripheral transcriptional responses to experimental SARS-CoV-2 inoculation in North American Elk cows and calves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 08:57:41","doi":"10.21203/rs.3.rs-6505643/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-27T18:39:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-25T18:29:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T20:27:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T15:35:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206611837204045856893229028407410904928","date":"2025-05-15T21:25:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24770253468282187270906884040132192180","date":"2025-05-15T20:58:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164414410787025793712991899443693750837","date":"2025-05-13T17:49:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T02:56:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-28T11:41:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-25T01:03:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T01:01:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-04-22T15:05:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"495a4cc6-84e7-48ea-810e-170dfdb6af7c","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:03:07+00:00","versionOfRecord":{"articleIdentity":"rs-6505643","link":"https://doi.org/10.1186/s12864-025-11956-5","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-08-28 15:58:08","publishedOnDateReadable":"August 28th, 2025"},"versionCreatedAt":"2025-05-15 08:57:41","video":"","vorDoi":"10.1186/s12864-025-11956-5","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11956-5","workflowStages":[]},"version":"v1","identity":"rs-6505643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6505643","identity":"rs-6505643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}