Transcriptomic Analysis Reveals Rnd1 as a Key Player in Antiviral Immunity Against Akabane virus via TNF-α pathway

Transcriptomic Analysis Reveals Rnd1 as a Key Player in Antiviral Immunity Against Akabane virus via TNF-α pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptomic Analysis Reveals Rnd1 as a Key Player in Antiviral Immunity Against Akabane virus via TNF-α pathway Dongjie Chen, Jingjing Wang, Chao Sun, Fang Wei, Shengkui Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6646858/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract Akabane virus (AKAV), the etiological agent of Akabane disease, is known to induce congenital malformations and neuropathologies in the fetuses of pregnant cattle and sheep. To comprehensively analyze the protein alterations and precisely elucidate the molecular mechanism in AKAV-susceptible cells, two types of primary bovine cells, namely primary bovine testicular sertoli cells (BTSC) and primary bovine joint synovial cells (BJSC), were selected. These cells were confirmed to be effectively infected by the AKAV TJ2016 strain. Subsequently, RNA-sequencing technology was employed to further analyze the transcriptomic profiles of AKAV-infected BTSC, BJSC, and MDOK cells. The molecular features of AKAV-infected cells demonstrated a remarkable activation of antiviral signaling pathways. Notably, there was an upregulation in the expression levels of interferon-stimulated genes, as well as genes related to inflammation and cytokines. Through a comparison between infected and non-infected cells, it was revealed that IL-1β, TNF-α, CXCL8, CCL2 and Rnd1 were significantly up-regulated in AKAV-infected cells. Moreover, Rnd1 was found to inhibit the replication of AKAV and TNF-αplays an important role in the induction of Rnd1, which provides additional evidence for the regulation and function of Rnd1. Akabane virus transcriptomic analysis Rnd1 TNF-α signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Akabane disease is an arthropod-borne viral disorder caused by Akabane virus (AKAV). This virus can infect cattle, sheep, and goats. In adult animals, it often causes inapparent clinical symptoms. However, in pregnant animals, it can lead to abortions, stillbirths, congenital arthrogryposis-hydranencephaly, and even death in offspring [ 1 , 2 ]. AKAV, the causative agent of Akabane disease, is an Arbovirus. It belongs to the genus Orthobunyavirus of the family Peribunyaviridae , order Elliovirales [ 3 ]. Similar to other members of its genus, the AKAV genome consists of three single-stranded negative-sense RNA segments, designated as large (L), medium (M), and small (S) segments. The L segment is responsible for encoding an RNA-dependent RNA polymerase (RdRp). The M segment encodes envelope glycoproteins Gn and Gc, which play a crucial role in facilitating the virus’s attachment to mammalian or insect cells. Additionally, the M segment encodes a non-structural NSm protein. The S segment encodes the nucleocapsid protein complex and a non-structural NSs protein. The NSs protein is known to suppress the host-cell antiviral response. Previous research has demonstrated that a suckling mouse model is widely utilized in the neurovirulence studies of AKAV [ 4 – 6 ]. This is because diverse AKAV strains can be detected within the central nervous system (CNS) of infected mice [ 7 , 8 ]. Experimentally, when injected into goats and cows, AKAV can induce encephalomyelitis with lesions observable in the parietal lobe of the cerebrum [ 9 , 10 ]. Notably, despite these findings, there has been relatively little exploration of other relevant tissues or cells in cows, particularly those potentially associated with the development of reproductive disorders. In the current study, we exposed different isolated bovine primary cells to AKAV. We verified that AKAV is capable of infecting both bovine testicular cells and bovine joint synovial cells. Subsequently, we conducted further transcriptomic analysis to compare protein expression profiles before and after infection. 2. Materials and Methods 2.1. Cells and Virus Madin-Darby Ovis Kidney (MDOK) cells were provided by Harbin Veterinary Research Institute. The primary bovine testicular sertoli cells (BTSC) and primary bovine joint synovial cells (BJSC) were isolated according to the references [ 11 , 12 ] and provided by Harbin Veterinary Research Institute. (Baby Hamster Kidney) BHK-21 cells were stored at Chinese Academy of Quality and Inspection & Testing (CAQI). All the above cells were maintained at 37℃ in an atmosphere of 5% CO 2 . AKAV TJ2016 was isolated and stored in CAQI according to the reference [ 13 ]. 2.2. Growth Kinetics of AKAV To determine the growth of AKAV in various cells types, multiple cell types were separately cultured in 24-well plates until they reached 80%-90% confluence. Cells were infected with AKAV TJ2016 at an multiplicity of infection (MOI) of 0.01. After a 1-hour (h) incubation, the unbound viruses were removed, and cells were washed three times with phosphate-buffered saline (PBS). Then, Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS; Gibco) was added. At specific time points post-infection, cells were harvested and the virus titers were determined using the tissue culture infectious dose 50 (TCID 50 ) method in BHK-21 cells, following the reference protocols [ 13 , 14 ]. 2.3. Indirect Immunofluorescence Assay For the immunofluorescence imaging of AKAV, cells were infected with the AKAV TJ2016 for 36 h and treated according to the reference [ 13 ]. Briefly, cells were fixed with 3.7% paraformaldehyde, and permeabilized with a solution of 0.1% triton X-100 in PBS containing 2% bovine serum albumin (BSA). Then, cells were incubated with primary antibodies. These included AKAV anti-N monoclonal antibody (diluted 1:1000, produced in CAQI), anti-PGP9.5 polyclonal antibody (diluted 1:1000, Cell Signaling Technology) and anti-FasL (diluted 1:1000, Abcam). Subsequently, the cells were stained with a FITC-conjugated goat anti-mouse secondary antibody (diluted 1:500, Solarbio Life Sciences). Finally, cell nuclei were stained with DAPI (4’6-diamidino-2-phenylindole). After five times washing, the plates were observed using the Invitrogen EVOS FL cell fluorescence imaging system (Thermo Fisher Scientific). 2.4. Sample Preparation, RNA Extraction, mRNA Library Construction and Sequencing For virus infection experiments, BTSC, BJSC and MDOK cells were incubated with AKAV TJ2016 at an MOI of 1 for 1 h, and washed by PBS to remove any unbound virus particles. Then, cells were cultured for 24 h, and the cell supernatants were discarded. Total RNA of the collected cells was then extracted using TRIzol reagent (Invitrogen). For the preparation of cDNA library construction, the total RNA samples were first quantified using a NanoDrop ND1000 spectrophotometer (Therm Fisher Scientific) and cDNA libraries were constructed following the standard illumina protocol, specifically using the NEBNext®Ultra TM II RNA Library Prep Kit for Illumina®. 2.5. Transcriptome Assembly and Transcriptional Profiling Analysis To annotate and assess the transcript abundances for the sequenced reads, the reference genomes of sheep or cattle, along with their annotations for protein-coding genes, were downloaded from the University of California at Santa Cruz ( http://genome.ucsc.edu ). After alignment, the HTSeq tool was employed to analyze the distribution of reads within known genes. Then, the Cuffdiff (v2.1.1) program was utilized to analyze the expression levels in BTSC, BJSC and MDOK cells infected with AKAV [ 15 ]. For differential gene expression analysis, Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems) was employed. Genes with a false discovery rate (FDR)-corrected p -value of less than 0.05 were identified as differentially expressed genes (DEGs) [ 16 ]. 2.6. Go Enrichment and KEGG Pathway Analysis Gene Ontology (GO) functional classification of DEGs were performed using Blast2GO software. The enriched gene functional categories were then analyzed. For pathway analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was accessed via the KOBAS software. A hypergeometric test was applied, and pathways with a corrected p -value < 0.05 were identified as significant [ 15 ]. 2.7. Quantitative Real-time Reverse Transcription-PCR (qRT-PCR) Total RNA was isolated from the samples using TRIzol reagent (Invitrogen), following the manufacturer’s protocol. Subsequently, the isolated RNA was reverse transcribed into cDNA using the reverse transcriptase kit (Tiangen). The resulting cDNA was then utilized for qRT-PCR analysis. The reactions were performed using the SYBR Green PCR master mix (Vazyme), and the reactions were run on a Light Cycler 480 instrument (Roche). To accurately determine the relative expression levels of the target mRNA, the expression data were normalized using the housekeeping gene GAPDH as an internal reference. The primers for the target genes (listed in Table 1 ) were carefully designed using Primer Premier 5 software. Table 1 primers used in the present study Name Sequences Rnd1 F: 5’-ACTCTGCTACAGCGACTCG−3’ R: 5’-CGGGTGCTGGGACAATAATC−3’ IL−1β F: 5’-CCCAAAAGTTACCCGAAGAGG−3’ R: 5’-TCTGCTTGAGAGGTGCTGATG−3’ TNF-ɑ F: 5’-ATGAGCACAGAAAGCATGA−3’ R: 5’-AGTAGACAGAAGAGCGTGGT−3’ AKAV-S F: 5’-CCACAACGGAATGCAGCTACAT−3’ R:5’-GTTGAGGAAGAAGACTCTAGCA−3’ CXCL8 F 5’-TCTGCAGCTCTGTGTGAAGGT−3’ R 5’-TGTGTTGGCGCAGTGTGGT−3’ CCL2 F 5’-TCTCCAGTCACCTGCTGCTA−3’ R 5’-TTTGGGTTTGGCTTTTCTTG−3’ GAPDH F: 5’-TGACTTCAACAGCGACACCCA−3’ R: 5’-CACCCTGTTGCTGTAGCCAAA−3’ TNF-ɑ siRNA Sense: 5’-GCCUACUGGCUGUGUACAU−3’ Antisense: 5’-AUGUACACAGCCAGUAGGC−3’ 2.8. Western Blot (WB) Analysis For WB analysis, 10 µg of whole cell lysates was subjected to separation via 12% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Following the electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The transferred PVDF membranes were blocked and subsequently probed with the corresponding primary antibodies specific to the target proteins. These primary antibodies recognize and bind to the proteins of interest on the membrane. After incubation with the primary antibodies, the membranes were washed three times with PBS containing 0.1% Tween 20 (PBST) and incubated with secondary antibodies, namely HRP-coupled goat anti-rabbit or anti-mouse antibodies, which were diluted at a ratio of 1:5000. Finally, membranes were thoroughly washed three times with PBST. The visualization of the target proteins was achieved by Immobilon™ Western HRP substrate peroxide solution (Millipore) and FluorChem E system (ProteinSimple) 2.9. Statistical Analysis All data in the present study were processed with GraphPad Prism 5 (GraphPad Software Inc.). The studen’s t-test was used to analyze the difference between the values of two groups. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. The Growth Curve of AKAV in BTSC and BJSC In the current study, the isolated BJSC and BTSC were cultured in six-well plates until they reached 80%-90% confluence. Subsequently, these cells were infected with AKAV TJ2016 strain at an MOI of 0.1. Thirty-six hours later, the cells were fixed. To identify the BJSC and BTSC, the PGP9.5 antibody and FasL antibody were used respectively, in accordance with previous research findings [12,17]. As depicted in Fig. 1A, the anti-PGP9.5 and anti-FasL antibodies labelled almost all of the isolated cells, indicating that AKAV was capable of efficiently infecting these isolated cells. Subsequently, BTSC, BJSC, MDOK and BHK-21 cells were infected with AKAV at an MOI of 0.1. The infected cells were collected at specific time points post-infection, namely 12 hours post-infection (hpi), 24 hpi, 36 hpi, and 48 hpi. And then the virus titers were measured using BHK-21 cells. As illustrated in Fig. 1B, and Fig. 1C, AKAV was able to infect and replicate effectively in both BTSC and BJSC. Notably, the replication efficiency of AKAV was higher in BTSC compared to BJSC, and both replication efficiencies were lower than those observed in MDOK and BHK-21 cells. 3.2 Different Expression Genes in Various AKAV Infected Cells To comprehensively understand the host response to AKAV infection, both mock-infected and AKAV-infected BTSC, BJSC or MDOK cells were harvested. Transcriptomic sequencing was then carried out using an Illumina HiSeq TM 2000 sequencer. Following sequencing, differential gene expression analysis was performed to identify DEGs between mock and AKAV-infected cells. DEGs were identified with a significance of p <0.05 and a fold change of at least 2. As shown in Fig. 2, at 24 hpi, 507 DEGs were detected in AKAV-infected BTSC—368 were upregulated and 139 were downregulated (Fig. 2A). In BJSC, the number of DEGs reached 1081, with 869 upregulated and 212 downregulated; in MDOK, 6626 DEGs were identified, with 3543 upregulated and 3083 downregulated (Fig. 2B and 2C). To identify common molecular responses across cell types, a Venn diagram analysis was conducted on the total DEGs of the three cell groups. This analysis revealed that 43 DEGs were upregulated or downregulated in all three cell types (Fig. 2D). Further details of the 43 DEGs were provided in Table 2. Among these DEGs, PTGS2, CXCR4, TNFAIP3, CXCL8, CCL2, CCL20, IL1A and Rnd1 were significantly associated with inflammatory signaling pathways. Specifically, these genes play important roles in mediating inflammatory responses, such as mediating immune cell recruitment and pro-inflammatory cytokine release. In addition, IRF1, as a key transcription factor, and GBP5, an IFN-stimulated gene product, were closely related to the interferon (IFN) signaling pathway, which are crucial for regulating antiviral responses and immune modulation via the IFN pathway. Table 2 Description of 43 common genes No. Name Description BTSC/log2Fc BJSC/log2Fc MDOK/log2Fc 1 PLEKHG4 pleckstrin homology and RhoGEF domain containing G4 -1.05 -1.45 -2.13 2 IRF1 interferon regulatory factor 1 1.33 2.10 3.45 3 PTGS2 prostaglandin-endoperoxide synthase 2 2.22 3.39 4.55 4 ZC3H12C zinc finger CCCH-type containing 12C 1.59 1.76 1.41 5 TAF4B TATA-box binding protein associated factor 4b 1.19 1.89 1.1 6 ETV3 ETS variant transcription factor 3 0.93 1.36 2.58 7 KIF1A kinesin family member 1A -2.25 -1.29 -1.33 8 BATF2 basic leucine zipper ATF-like transcription factor 2 1.19 7.37 6.97 9 C1R complement C1r -1.04 1.56 1.02 10 KIF26A kinesin family member 26A 1.89 5.41 -1.82 11 BHLHE41 basic helix-loop-helix family member e41 1.54 1.67 4.15 12 CXCR4 C-X-C motif chemokine receptor 4 1.89 3.67 3.39 13 TNFAIP3 TNF alpha induced protein 3 3.24 4.40 5.16 14 CXCL8 C-X-C motif chemokine ligand 8 1.78 4.03 8.63 15 TNFA TNF alpha 1.31 3.56 2.32 16 DUSP10 dual specificity phosphatase 10 1.02 1.79 1.48 17 TIPARP TCDD inducible poly(ADP-ribose) polymerase 1.58 1.27 2.13 18 CSRNP1 cysteine and serine rich nuclear protein 1 1.67 1.86 3.33 19 NFKBIA NFKB inhibitor alpha 2.20 2.65 4.91 20 NFKBIE NFKB inhibitor epsilon 1.26 1.46 1.62 21 ZFP36 ZFP36 ring finger protein 0.70 0.44 1.29 22 CGNL1 cingulin like 1 2.02 2.76 1.03 23 FBXO33 F-box protein 33 1.01 3.01 1.06 24 CD274 CD274 molecule 1.22 4.41 6.38 25 SOCS2 suppressor of cytokine signaling 2 1.44 1.40 2.11 26 CCL2 chemokine (C-C motif) ligand 2 1.04 3.67 3.86 27 TANC2 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 1.82 1.38 2.32 28 TNFAIP2 TNF alpha induced protein 2 2.45 1.53 3.81 29 CD40 CD40 molecule 1.27 2.42 2.48 30 BIRC3 baculoviral IAP repeat containing 3 1.34 2.50 3.45 31 GBP5 guanylate binding protein 5 1.17 5.12 7.19 32 CCL20 C-C motif chemokine ligand 20 3.0 6.49 8.49 33 SYT12 synaptotagmin 12 -2.10 -1.23 -1.97 34 ZMYND10 zinc finger MYND-type containing 10 1.47 2.31 2.44 35 IL1B interleukin 1 beta 1.99 2.82 1.41 36 RND1 Rho family GTPase 1 3.95 3.75 4.68 37 FAM171A2 family with sequence similarity 171 member A2 -2.33 -1.66 -2.86 38 SEMA6C semaphorin 6C 1.50 -1.36 -2.23 39 SH3RF2 SH3 domain containing ring finger 2 1.73 3.29 1.91 40 KIF21B kinesin family member 21B -1.16 -1.46 -2.49 41 LY6E lymphocyte antigen 6 family member E -1.59 1.37 -3.14 42 HES4 hes family bHLH transcription factor 4 1.18 6.94 1.53 43 PRR22 proline rich 22 -6.15 -5.27 -2.46 3.3 Functional Enrichment Analysis of DEGs in Various Groups To gain deeper insights into the functions of DEGs between AKAV-infected and control cells, and to enhance our understanding of the role of host proteins in generating antiviral factors during AKAV infection, we conducted GO and KEGG enrichment analyses. GO enrichment analysis is a widely adopted approach for revealing the relationships between genes and functional terms. KEGG enrichment analysis, on the other hand, effectively illustrates the associations between genes and biological pathways. As depicted in Figure 3A and 3B, GO enrichment analysis of AKAV-infected BJSC and BTSC demonstrated significant enrichment in several biological processes (BPs). These included cytokine-mediated signaling pathwasy, specifically involving IL-1β and IL-1, and antiviral-related pathways such as the response to exogenous dsRNA, response to IFN-β, and regulation of the mitogen-activated protein kinase (MAPK) cascade. In contrast, in AKAV-infected MDOK cells, BP enrichment was primarily observed in the regulation of the MAPK cascade, protein localization to the plasma membrane, and extracellular matrix organization (Fig. 3C). KEGG enrichment analysis further indicated that DEGs were predominantly involved in pathways such as the TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, NOD-like receptor pathway, and herpes simplex virus 1 infection (Fig. 3D－F). The above results indicate that AKAV infection can induce the activation of inflammatory- and antiviral-associated pathways. 3.4 Interferon and Inflammtory Responses are Activated in AKAV-infected Cells We demonstrated that AKAV infection induced substantial alterations in the transcriptomic profiles of host cells. To further validate these changes, we focused on interferon- and inflammation-associated cytokines. Using reverse transcription polymerase chain reaction (RT-PCR), we quantified the transcript levels of TNF-α, IL-1β, CXCL8, CCL2, and Rnd1 in AKAV-infected cells. The fold changes in gene expression were calculated using the 2-ΔΔCt method. For normalization, we used the Ct values of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control for each sample. The results revealed that AKAV infection upregulated the expression of TNF-α, IL-1β, CXCL8, CCL2, and Rnd1 in all BTSC, BJSC, and MDOK cells (Fig. 4), which was in correspondence with the transcriptomic results. 3.5 AKAV Induced Rnd1 Overexpression Through TNF- α Signaling Pathway Since Rnd1 expression was significantly upregulated in all AKAV-infected cells described above, we delved deeper into exploring its function and mechanism. MDOK cells were transfected with either the Rnd1-encoding plasmid (pCMV-Flag-Rnd1) or the vector backbone (pCMV-Flag-6C). After transfection, the cells were infected with AKAV. To determine the viral load, we employed two methods: the TCID 50 assay and WB. Analyses were carried out at 12 hpi and 24 hpi. As depicted in Fig. 5, after AKAV infection, transfection of MDOK cells with pCMV-Flag-Rnd1 resulted in a substantial reduction in AKAV viral load. This was evident in both the TCID 50 assay and WB analysis, when compared to control cells transfected with the vector backbone. To further investigate the role of TNF-α in AKAV-induced Rnd1 overexpression, we employed two experimental approaches. Ten ng/mL of recombinant TNF-α (purchased from MCE, China) was added to the cell culture, or TNF-α-targeting siRNA (Table 1) was transfected into cells. Twelve hours later, cells were infected with AKAV at an MOI of 0.1, and the viral load was detected in 12 hpi and 24 hpi by TCID 50 and WB. As depicted in Fig. 5B, the addition of TNF-α significantly upregulated Rnd1 expression. Concomitantly, there was a reduction in AKAV viral load. Conversely, when TNF-α expression was silenced using siRNA, Rnd1 expression decreased. This was accompanied by an upregulation of AKAV replication (Fig. 5C). These results suggest that TNF-α plays a crucial role in modulating Rnd1 expression and, subsequently, influences the replication of AKAV within host cells. 4. Discussion As one member of bunyavirus, AKAV is a Culicoides-borne virus and is teratogenic to the fetus of cattle and small ruminant species. When the cow or sheep was infected with AKAV during pregnancy, the adult cattle generally do not show clinical symptoms, while the fetus may develop with congenital malformations, including hydranencephaly, poliomyelitis and arthrogryposis [1]. Previous research has shown that AKAV can cross the placenta, and penetrate the blood-fetal barrier. This transplacental transmission leads to restricted reproductive disorders, resulting in congenital abnormalities [18]. However, the precise molecular mechanisms underpinning these processes remain unclear. Additionally, it is unknown whether AKAV can trigger reproductive disorders through alternative routes, such as via testicular cells. In this study, we systematically analyzed the infectivity of AKAV in multiple primary bovine cells. These included bovine turbinate cells, bovine duct epithelial cells, bovine lung epithelial cells, bovine mammary epithelial cells, BTSC, and BJSC. Our results demonstrate that AKAV could replicate efficiently in BTSC and BJSC. In contrast, only a small percentage (around 20%, data not shown) of bovine turbinate cells were infected. Through transcriptomic analysis, we found that AKAV infection significantly upregulates the expression of inflammatory-related genes, including TNF-α, IL-1β, CXCL8, and CCL2, which can partly explain why AKAV can cause reproductive disorders and joint related lesions[19]. The Rho family of GTPase consists of 20 members. In humans, these members are broadly classified into classic and atypical groups [20]. Classic Rho GTPases, prominently represented by RHOA, CDC42, and RAC1, function as molecular switches. They cycle between an inactive state, bound to DiPhosphate (GDP), and an active state, bound to Guanosine TriPhosphate (GTP) [21]. Given their significance in various cellular processes, classic Rho GTPase family members have been extensively studied. In contrast, atypical molecules such as the Rnd family proteins have received relatively less attention. Rnd1, also known as RHO6, is an atypical Rho GTPase. Rnd1 belongs to the RND subfamily, which also includes members Rnd2 (RHO7/RHON) and Rnd3 (RHO8/RHOE). All RND proteins feature the Rho-specific insert domain, a characteristic defining the Rho GTPase family. At the N-terminal region, Rnd1 and Rnd3 (but not Rnd2) possess a KERRA (Lys-Glu-Arg-Arg-Ala) sequence. This sequence facilitates their targeting to lipid rafts, thereby determining their localization at the plasma membrane [22]. Accumulating evidence from previous studies demonstrates that RNDs play crucial roles in axon guidance, cell cycle regulation, and tumorigenesis [23,24]. However, their relationship with virus infection remains largely unexplored. Previous study reported that Rnd1 can be induced by pro-inflammatory cytokines during viral and bacterial infections and provides protection against these pathogens through two distinct mechanisms [25]. First, Rnd1 acts as a regulator of intracellular calcium homeostasis, which inhibits RhoA activation to counter calcium fluctuations and block virus entry. Also, Rnd1 facilitates pro-inflammatory cytokines IL-6 and TNF-α through Plxnb1, which are highly effective against intracellular bacterial infections. However, in another study, TNF-α, IL-1β, and IL-6 also increased in rheumatoid synovial cells via Rnd1 overexpression [26,27]. In our study, we determined that following AKAV infection, the expression levels of Rnd1, IL-6, and TNF-α were significantly upregulated. TNF-α appears to play a crucial role in the overexpression of Rnd1. However, the precise molecular mechanisms underlying this regulatory relationship remain unclear and need further investigation. 5. Conclusions In summary, we utilized primary BTSC and BJSC to evaluate the infectivity of AKAV, confirming that both cell types are susceptible to AKAV TJ2016 strain. Through transcriptomic analysis, we found that AKAV infection significantly up-regulated interferon-stimulated genes, inflammation-related genes, and cytokines. Notably, we demonstrated that Rnd1 was significantly elevated in infected cells and showed that Rnd1 inhibits AKAV replication. Additionally, TNF-α was identified as a key inducer of Rnd1, providing mechanistic insights into antiviral regulation during AKAV infection. Declarations Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Author contributions Dong-jie Chen: analysed data, drafted the manuscript and performed the laboratory experiments. Jing-jing Wang: analysed data and performed the laboratory experiments. Chao Sun: collected bovine primary cells. Fang Wei: collected data. Sheng-kui Xu: performed the laboratory experiments. Ru-yang Yu: analysed data. Shao-qiang Wu: supervised the study and finalized the manuscript. Declaration of competing interest The authors of this study declared that they do not have any conflict of interest. Acknowledgments This work was supported by the National Key R&D Program of China (2022YFD1802000), and the Natural Science Foundation of Beijing (6254044). 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Briefings Bioinform 20(6): 2044–2054. https://doi.org/10.1093/bib/bby067 LinYC, Lu M, Cai W, Hu WS (2013) Comparative transcriptomic and proteomic kinetic analysis of adeno-associated virus production systems. Appl Microbiol Biotechnol 108,08(1): 385. https://doi.org/10.1007/s00253-024-13203-51 Wang G, Wang Y, Kong J, Li Y, Wu J, Chen Y, Liu X, Shang Y, Zhang Z (2019) Comparison of the sensitivity of three cell cultures to ORFV. BMC Vet Res 15(1):13. https://doi.org/10.1186/s12917-018-1760-1 Parsonson IM, Della - Porta AJ, Snowdon WA (1977) Congenital abnormalities in newborn lambs after infection of pregnant sheep with Akabane virus. Infect Immun 15(1):254–262. https://doi.org/10.1128/iai.15.1.254-262.1977 Matumoto M, Inaba Y. (1980) Akabane disease and Akabane virus. Kitasato Arch Exp Med 53(1-2): 1–21. Mosaddeghzadeh N, Ahmadian MR (2021) The RHO Family GTPases: Mechanisms of Regulation and Signaling. Cells 10(7):1831. https://doi.org/10.3390/cells10071831 Bagci H, Sriskandarajah N, Robert A, Boulais J, Elkholi IE, Tran V, Lin ZY, Thibault MP, Dubé N, Faubert D, Hipfner DR, Gingras AC, Côté JF (2020) Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms. Nat Cell Biol 22(1):120–134. https://doi.org/10.1038/s41556-019-0438-7 Oinuma I, Kawada K, Tsukagoshi K, Negishi M (2012) Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation. Mol Biol Cell 23(8):1593–1604. https://doi.org/10.1091/mbc.E11-11-0900 Sun Q, Xu J, Yuan F, Liu Y, Chen Q, Guo L, Dong H, Liu B (2024) RND1 inhibits epithelial-mesenchymal transition and temozolomide resistance of glioblastoma via AKT/GSK3-β pathway. Cancer Biol Ther 25(1):2321770. https://doi.org/10.1080/15384047.2024.2321770 Mouly L, Gilhodes J, Lemarié A, Cohen-Jonathan Moyal E, Toulas C, Favre G, Sordet O, Monferran S (2019) The RND1 small GTPase: Main functions and emerging role in oncogenesis. Int J Mol Sci 20(15):3612. https://doi.org/10.3390/ijms20153612 Kumar A, Mishra S, Kumar A, Raut AA, Sato S, Takaoka A, Kumar H (2022) Essential role of Rnd1 in innate immunity during viral and bacterial infections. Cell death & disease, 13(6), 520. https://doi.org/10.1038/s41419-022-04954-y Chen Q, Chen D, Wang S, Huang X, Liang L, Xie T, Lu J (2025) RND1 induces ferroptosis to alleviate inflammatory response, proliferation, invasion, and migration of rheumatoid synoviocytes. J Inflamm Res 18:2647–2659. https://doi.org/10.2147/JIR.S500630 Möller B, Villiger PM (2006) Inhibition of IL-1, IL-6, and TNF-alpha in immune-mediated inflammatory diseases. Springer Semin Immunopathol 27(4):391–408. https://doi.org/10.1007/s00281-006-0012-9 Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 13 Jun, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 27 May, 2025 First submitted to journal 27 May, 2025 Editorial decision: Major Revision 15 May, 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. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6646858","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470697599,"identity":"5190e74a-f260-499d-a158-317c95db31a8","order_by":0,"name":"Dongjie Chen","email":"","orcid":"","institution":"Institute of Animal Quarantine and Inspection, Chinese Academy of Quality and Inspection \u0026 Testing,","correspondingAuthor":false,"prefix":"","firstName":"Dongjie","middleName":"","lastName":"Chen","suffix":""},{"id":470697600,"identity":"487c6852-3cc1-4208-b032-b200a793b65f","order_by":1,"name":"Jingjing Wang","email":"","orcid":"","institution":"Institute of Animal Quarantine and Inspection, Chinese Academy of Quality and Inspection \u0026 Testing,","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Wang","suffix":""},{"id":470697601,"identity":"6d833780-9dfb-488f-8270-dddcb5df467d","order_by":2,"name":"Chao Sun","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences Harbin Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Sun","suffix":""},{"id":470697602,"identity":"94e49696-86ed-4736-bfa9-e167b0707693","order_by":3,"name":"Fang Wei","email":"","orcid":"","institution":"Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Wei","suffix":""},{"id":470697603,"identity":"baf27a61-4e2a-4187-9ba1-f4af76c094a1","order_by":4,"name":"Shengkui Xu","email":"","orcid":"","institution":"Beijing University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Shengkui","middleName":"","lastName":"Xu","suffix":""},{"id":470697604,"identity":"4730e111-8435-41cb-969b-6ec66788a2b3","order_by":5,"name":"Ruyang Yu","email":"","orcid":"","institution":"Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Ruyang","middleName":"","lastName":"Yu","suffix":""},{"id":470697605,"identity":"edf09c38-3aff-4d68-9265-bffa56d0eda1","order_by":6,"name":"Shaoqiang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAm0lEQVRIiWNgGAWjYLCCDwwSJOpgnEGyFmYekpTLt+eYSdv8scjjb2B++OgGMVoMzrwxk85tkyiWOMBmbJxDlBaJ3G3SuQ0SiQ0HeNikidIiPwOoxeKPROJ8orUw3ABqYWCTSNxAtBaDM+8/W/a2SSRuPEysX+Tb0xJv/PhTlzjvePPDx8Q5jCEBSjMTpxxZyygYBaNgFIwCXAAAgmwtfVT5l2gAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3255-8020","institution":"Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":true,"prefix":"","firstName":"Shaoqiang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-05-12 12:56:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6646858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6646858/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06477-1","type":"published","date":"2025-11-28T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84820226,"identity":"6fabb1bd-0ac0-4cae-a825-6d014f312a99","added_by":"auto","created_at":"2025-06-17 16:06:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":361732,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of AKAV replication in BJSC, BTSC, MDOK and BHK-21 cells.\u003cstrong\u003e A\u003c/strong\u003e Detection of AKAV infection and characterization of BJSC and BTSC; \u003cstrong\u003eB\u003c/strong\u003e Determination of AKAV proliferation curves in BJSC, BTSC, MDOK and BHK-21 cells; C WB analysis of AKAV in BJSC, BTSC, MDOK and BHK-21 cells.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/d83f291df3bd1105ecc46eb8.jpg"},{"id":84820232,"identity":"a40cc95d-99b8-4f2f-98d7-a7f557bf5c02","added_by":"auto","created_at":"2025-06-17 16:06:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247210,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs of AKAV infection in BTSC, BJSC and MDOK cells. \u003cstrong\u003eA \u003c/strong\u003eDEGs of AKAV infection in BTSC; \u003cstrong\u003eB\u003c/strong\u003e DEGs of AKAV infection in BJSC;\u003cstrong\u003e C\u003c/strong\u003e DEGs of AKAV infection in MDOK; D Venn diagram analysis of DEGs among different AKAV infected groups.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/8f9b5b4c77dc8692788e4b95.jpg"},{"id":84821815,"identity":"2a749f61-656a-49ee-ab58-1fe48347106b","added_by":"auto","created_at":"2025-06-17 16:14:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":528811,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analysis of DEGs. \u003cstrong\u003eA\u003c/strong\u003eGO enrichment analysis of DEGs in BTSC and Control; \u003cstrong\u003eB\u003c/strong\u003e GO enrichment analysis of DEGs in BJSC and Control; \u003cstrong\u003eC\u003c/strong\u003e GO enrichment analysis of DEGs in MDOK and Control; \u003cstrong\u003eD\u003c/strong\u003e KEGG enrichment analysis of DEGs in BTSC and Control; \u003cstrong\u003eE\u003c/strong\u003e KEGG enrichment analysis of DEGs in BJSC and Control; \u003cstrong\u003eF\u003c/strong\u003eKEGG enrichment analysis of DEGs in MDOK and Control. Y-axis represents Go term or KEGG pathway, and X-axis represents Rich factor.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/6e7ee09c6b963be4e822588c.jpg"},{"id":84821818,"identity":"6cdd49db-1d10-43c9-ad6b-cfae4bce9e4f","added_by":"auto","created_at":"2025-06-17 16:14:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209238,"visible":true,"origin":"","legend":"\u003cp\u003eRT-qPCR verification of five genes. We examined the gene expression levels of TNF-α, IL-1β, Rnd1, CXCL8 and CCL2. \u003cstrong\u003eA\u003c/strong\u003e RT-qPCR verification of TNF-α; \u003cstrong\u003eB\u003c/strong\u003e RT-qPCR verification of CXCL8; \u003cstrong\u003eC\u003c/strong\u003eRT-qPCR verification of IL-1β; \u003cstrong\u003eD\u003c/strong\u003e RT-qPCR verification of CCL2; \u003cstrong\u003eE\u003c/strong\u003eRT-qPCR verification of Rnd1. The error bars indicate the SD of repeated RT-qPCR. All experiments were conducted independently, at least three times.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/9ee928e2031ba49e4b5527f9.jpg"},{"id":84820229,"identity":"1fa4c805-9c06-4143-aa2c-d59fa50c02fb","added_by":"auto","created_at":"2025-06-17 16:06:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":188960,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α induced Rnd1 Overexpression inhibits the replication of AKAV . \u003cstrong\u003eA\u003c/strong\u003e MDOK cells were transfected with either empty vector backbone or Rnd1 expressing plasmids followed by infection with AKAV at an MOI of 0.1. Twelve and 24 h later, the replication of AKAV was detected by WB and TCID\u003csub\u003e50\u003c/sub\u003e; \u003cstrong\u003eB\u003c/strong\u003e MDOK cells were pretreated with 10 ng/mL TNF-α, and infected with AKAV at an MOI of 0.1. Twelve and 24 h later, the replication of AKAV was detected by WB and TCID\u003csub\u003e50\u003c/sub\u003e; \u003cstrong\u003eC\u003c/strong\u003e MDOK cells were transfected with siRNA of TNF-α, and infected with AKAV at an MOI of 0.1. Twelve and 24 h later, the replication of AKAV was detected by WB and TCID\u003csub\u003e50.\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/f600f7fffd788f6c5c23cd13.jpg"},{"id":97178222,"identity":"36bb325e-cbe0-40e4-bbdc-6a8ac6b2c916","added_by":"auto","created_at":"2025-12-01 16:01:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2324845,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6646858/v1/a4c03db7-90e8-47ec-9fc7-95452dddb5d8.pdf"}],"financialInterests":"","formattedTitle":"Transcriptomic Analysis Reveals Rnd1 as a Key Player in Antiviral Immunity Against Akabane virus via TNF-α pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAkabane disease is an arthropod-borne viral disorder caused by Akabane virus (AKAV). This virus can infect cattle, sheep, and goats. In adult animals, it often causes inapparent clinical symptoms. However, in pregnant animals, it can lead to abortions, stillbirths, congenital arthrogryposis-hydranencephaly, and even death in offspring [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. AKAV, the causative agent of Akabane disease, is an Arbovirus. It belongs to the genus \u003cem\u003eOrthobunyavirus\u003c/em\u003e of the family \u003cem\u003ePeribunyaviridae\u003c/em\u003e, order \u003cem\u003eElliovirales\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Similar to other members of its genus, the AKAV genome consists of three single-stranded negative-sense RNA segments, designated as large (L), medium (M), and small (S) segments. The L segment is responsible for encoding an RNA-dependent RNA polymerase (RdRp). The M segment encodes envelope glycoproteins Gn and Gc, which play a crucial role in facilitating the virus\u0026rsquo;s attachment to mammalian or insect cells. Additionally, the M segment encodes a non-structural NSm protein. The S segment encodes the nucleocapsid protein complex and a non-structural NSs protein. The NSs protein is known to suppress the host-cell antiviral response.\u003c/p\u003e \u003cp\u003ePrevious research has demonstrated that a suckling mouse model is widely utilized in the neurovirulence studies of AKAV [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This is because diverse AKAV strains can be detected within the central nervous system (CNS) of infected mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Experimentally, when injected into goats and cows, AKAV can induce encephalomyelitis with lesions observable in the parietal lobe of the cerebrum [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, despite these findings, there has been relatively little exploration of other relevant tissues or cells in cows, particularly those potentially associated with the development of reproductive disorders. In the current study, we exposed different isolated bovine primary cells to AKAV. We verified that AKAV is capable of infecting both bovine testicular cells and bovine joint synovial cells. Subsequently, we conducted further transcriptomic analysis to compare protein expression profiles before and after infection.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Cells and Virus\u003c/h2\u003e\n \u003cp\u003eMadin-Darby Ovis Kidney (MDOK) cells were provided by Harbin Veterinary Research Institute. The primary bovine testicular sertoli cells (BTSC) and primary bovine joint synovial cells (BJSC) were isolated according to the references [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e] and provided by Harbin Veterinary Research Institute. (Baby Hamster Kidney) BHK-21 cells were stored at Chinese Academy of Quality and Inspection \u0026amp; Testing (CAQI). All the above cells were maintained at 37℃ in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. AKAV TJ2016 was isolated and stored in CAQI according to the reference [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Growth Kinetics of AKAV\u003c/h2\u003e\n \u003cp\u003eTo determine the growth of AKAV in various cells types, multiple cell types were separately cultured in 24-well plates until they reached 80%-90% confluence. Cells were infected with AKAV TJ2016 at an multiplicity of infection (MOI) of 0.01. After a 1-hour (h) incubation, the unbound viruses were removed, and cells were washed three times with phosphate-buffered saline (PBS). Then, Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS; Gibco) was added. At specific time points post-infection, cells were harvested and the virus titers were determined using the tissue culture infectious dose 50 (TCID\u003csub\u003e50\u003c/sub\u003e) method in BHK-21 cells, following the reference protocols [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Indirect Immunofluorescence Assay\u003c/h2\u003e\n \u003cp\u003eFor the immunofluorescence imaging of AKAV, cells were infected with the AKAV TJ2016 for 36 h and treated according to the reference [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, cells were fixed with 3.7% paraformaldehyde, and permeabilized with a solution of 0.1% triton X-100 in PBS containing 2% bovine serum albumin (BSA). Then, cells were incubated with primary antibodies. These included AKAV anti-N monoclonal antibody (diluted 1:1000, produced in CAQI), anti-PGP9.5 polyclonal antibody (diluted 1:1000, Cell Signaling Technology) and anti-FasL (diluted 1:1000, Abcam). Subsequently, the cells were stained with a FITC-conjugated goat anti-mouse secondary antibody (diluted 1:500, Solarbio Life Sciences). Finally, cell nuclei were stained with DAPI (4\u0026rsquo;6-diamidino-2-phenylindole). After five times washing, the plates were observed using the Invitrogen EVOS FL cell fluorescence imaging system (Thermo Fisher Scientific).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Sample Preparation, RNA Extraction, mRNA Library Construction and Sequencing\u003c/h2\u003e\n \u003cp\u003eFor virus infection experiments, BTSC, BJSC and MDOK cells were incubated with AKAV TJ2016 at an MOI of 1 for 1 h, and washed by PBS to remove any unbound virus particles. Then, cells were cultured for 24 h, and the cell supernatants were discarded. Total RNA of the collected cells was then extracted using TRIzol reagent (Invitrogen). For the preparation of cDNA library construction, the total RNA samples were first quantified using a NanoDrop ND1000 spectrophotometer (Therm Fisher Scientific) and cDNA libraries were constructed following the standard illumina protocol, specifically using the NEBNext\u0026reg;Ultra\u003csup\u003eTM\u003c/sup\u003eII RNA Library Prep Kit for Illumina\u0026reg;.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Transcriptome Assembly and Transcriptional Profiling Analysis\u003c/h2\u003e\n \u003cp\u003eTo annotate and assess the transcript abundances for the sequenced reads, the reference genomes of sheep or cattle, along with their annotations for protein-coding genes, were downloaded from the University of California at Santa Cruz (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genome.ucsc.edu\u003c/span\u003e\u003c/span\u003e). After alignment, the HTSeq tool was employed to analyze the distribution of reads within known genes. Then, the Cuffdiff (v2.1.1) program was utilized to analyze the expression levels in BTSC, BJSC and MDOK cells infected with AKAV [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. For differential gene expression analysis, Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems) was employed. Genes with a false discovery rate (FDR)-corrected \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 were identified as differentially expressed genes (DEGs) [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Go Enrichment and KEGG Pathway Analysis\u003c/h2\u003e\n \u003cp\u003eGene Ontology (GO) functional classification of DEGs were performed using Blast2GO software. The enriched gene functional categories were then analyzed. For pathway analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was accessed via the KOBAS software. A hypergeometric test was applied, and pathways with a corrected \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as significant [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Quantitative Real-time Reverse Transcription-PCR (qRT-PCR)\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated from the samples using TRIzol reagent (Invitrogen), following the manufacturer\u0026rsquo;s protocol. Subsequently, the isolated RNA was reverse transcribed into cDNA using the reverse transcriptase kit (Tiangen). The resulting cDNA was then utilized for qRT-PCR analysis. The reactions were performed using the SYBR Green PCR master mix (Vazyme), and the reactions were run on a Light Cycler 480 instrument (Roche). To accurately determine the relative expression levels of the target mRNA, the expression data were normalized using the housekeeping gene GAPDH as an internal reference. The primers for the target genes (listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) were carefully designed using Primer Premier 5 software.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eprimers used in the present study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRnd1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-ACTCTGCTACAGCGACTCG\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR: 5\u0026rsquo;-CGGGTGCTGGGACAATAATC\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIL\u0026minus;1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-CCCAAAAGTTACCCGAAGAGG\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR: 5\u0026rsquo;-TCTGCTTGAGAGGTGCTGATG\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTNF-ɑ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-ATGAGCACAGAAAGCATGA\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR: 5\u0026rsquo;-AGTAGACAGAAGAGCGTGGT\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAKAV-S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-CCACAACGGAATGCAGCTACAT\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR:5\u0026rsquo;-GTTGAGGAAGAAGACTCTAGCA\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCXCL8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF 5\u0026rsquo;-TCTGCAGCTCTGTGTGAAGGT\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR 5\u0026rsquo;-TGTGTTGGCGCAGTGTGGT\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF 5\u0026rsquo;-TCTCCAGTCACCTGCTGCTA\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR 5\u0026rsquo;-TTTGGGTTTGGCTTTTCTTG\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-TGACTTCAACAGCGACACCCA\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR: 5\u0026rsquo;-CACCCTGTTGCTGTAGCCAAA\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTNF-ɑ siRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSense: 5\u0026rsquo;-GCCUACUGGCUGUGUACAU\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntisense: 5\u0026rsquo;-AUGUACACAGCCAGUAGGC\u0026minus;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Western Blot (WB) Analysis\u003c/h2\u003e\n \u003cp\u003eFor WB analysis, 10 \u0026micro;g of whole cell lysates was subjected to separation via 12% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Following the electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The transferred PVDF membranes were blocked and subsequently probed with the corresponding primary antibodies specific to the target proteins. These primary antibodies recognize and bind to the proteins of interest on the membrane. After incubation with the primary antibodies, the membranes were washed three times with PBS containing 0.1% Tween 20 (PBST) and incubated with secondary antibodies, namely HRP-coupled goat anti-rabbit or anti-mouse antibodies, which were diluted at a ratio of 1:5000. Finally, membranes were thoroughly washed three times with PBST. The visualization of the target proteins was achieved by Immobilon\u0026trade; Western HRP substrate peroxide solution (Millipore) and FluorChem E system (ProteinSimple)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data in the present study were processed with GraphPad Prism 5 (GraphPad Software Inc.). The studen\u0026rsquo;s t-test was used to analyze the difference between the values of two groups. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1. The Growth Curve of AKAV in BTSC and BJSC\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the current study, the isolated BJSC and BTSC were cultured in six-well plates until they reached 80%-90% confluence. Subsequently, these cells were infected with AKAV TJ2016 strain at an MOI of 0.1. Thirty-six hours later, the cells were fixed. To identify the BJSC and BTSC, the PGP9.5 antibody and FasL antibody were used respectively, in accordance with previous research findings [12,17]. As depicted in Fig. 1A, the anti-PGP9.5 and anti-FasL antibodies labelled almost all of the isolated cells, indicating that AKAV was capable of efficiently infecting these isolated cells. Subsequently, BTSC, BJSC, MDOK and BHK-21 cells were infected with AKAV at an MOI of 0.1. The infected cells were collected at specific time points post-infection, namely 12 hours post-infection (hpi), 24 hpi, 36 hpi, and 48 hpi. And then the virus titers were measured using BHK-21 cells. As illustrated in Fig. 1B, and Fig. 1C, AKAV was able to infect and replicate effectively in both BTSC and BJSC. Notably, the replication efficiency of AKAV was higher in BTSC compared to BJSC, and both replication efficiencies were lower than those observed in MDOK and BHK-21 cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 Different Expression Genes in Various AKAV Infected Cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively understand the host response to AKAV infection, both mock-infected and AKAV-infected BTSC, BJSC or MDOK cells were harvested. Transcriptomic sequencing was then carried out using an Illumina HiSeq\u003csup\u003eTM\u003c/sup\u003e2000 sequencer. Following sequencing, differential gene expression analysis was performed to identify DEGs between mock and AKAV-infected cells. DEGs were identified with a significance of \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and a fold change of at least 2. As shown in Fig. 2, at 24 hpi, 507 DEGs were detected in AKAV-infected BTSC\u0026mdash;368 were upregulated and 139 were downregulated (Fig. 2A). In BJSC, the number of DEGs reached 1081, with 869 upregulated and 212 downregulated; in MDOK, 6626 DEGs were identified, with 3543 upregulated and 3083 downregulated (Fig. 2B and 2C). To identify common molecular responses across cell types, a Venn diagram analysis was conducted on the total DEGs of the three cell groups. This analysis revealed that 43 DEGs were upregulated or downregulated in all three cell types (Fig. 2D). Further details of the 43 DEGs were provided in Table 2. Among these DEGs, PTGS2, CXCR4, TNFAIP3, CXCL8, CCL2, CCL20, IL1A and Rnd1 were significantly associated with inflammatory signaling pathways. Specifically, these genes play important roles in mediating inflammatory responses, such as mediating immune cell recruitment and pro-inflammatory cytokine release. In addition, IRF1, as a key transcription factor, and GBP5, an IFN-stimulated gene product, were closely related to the interferon (IFN) signaling pathway, which are crucial for regulating antiviral responses and immune modulation via the IFN pathway.\u003c/p\u003e\n\u003cp\u003eTable 2 Description of 43 common genes\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"651\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eBTSC/log2Fc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003eBJSC/log2Fc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMDOK/log2Fc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003ePLEKHG4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003epleckstrin homology and RhoGEF domain containing G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eIRF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003einterferon regulatory factor 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003ePTGS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eprostaglandin-endoperoxide synthase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eZC3H12C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ezinc finger CCCH-type containing 12C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTAF4B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTATA-box binding protein associated factor 4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eETV3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eETS variant transcription factor 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eKIF1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ekinesin family member 1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eBATF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ebasic leucine zipper ATF-like transcription factor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e7.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e6.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eC1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ecomplement C1r\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eKIF26A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ekinesin family member 26A\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e5.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eBHLHE41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ebasic helix-loop-helix family member e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCXCR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eC-X-C motif chemokine receptor 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTNFAIP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTNF alpha induced protein 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e4.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCXCL8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eC-X-C motif chemokine ligand 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e4.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e8.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTNFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTNF alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eDUSP10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003edual specificity phosphatase 10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTIPARP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTCDD inducible poly(ADP-ribose) polymerase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCSRNP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ecysteine and serine rich nuclear protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eNFKBIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eNFKB inhibitor alpha\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eNFKBIE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eNFKB inhibitor epsilon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eZFP36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eZFP36 ring finger protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCGNL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ecingulin like 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eFBXO33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eF-box protein 33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCD274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eCD274 molecule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e4.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e6.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eSOCS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003esuppressor of cytokine signaling 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003echemokine (C-C motif) ligand 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTANC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003etetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eTNFAIP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eTNF alpha induced protein 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCD40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eCD40 molecule\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eBIRC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ebaculoviral IAP repeat containing 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eGBP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eguanylate binding protein 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e5.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e7.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eCCL20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eC-C motif chemokine ligand 20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e6.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e8.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eSYT12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003esynaptotagmin 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eZMYND10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ezinc finger MYND-type containing 10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eIL1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003einterleukin 1 beta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eRND1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eRho family GTPase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eFAM171A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003efamily with sequence similarity 171 member A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-2.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eSEMA6C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003esemaphorin 6C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eSH3RF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eSH3 domain containing ring finger 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e3.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eKIF21B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ekinesin family member 21B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eLY6E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003elymphocyte antigen 6 family member E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-3.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eHES4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003ehes family bHLH transcription factor 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e6.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003ePRR22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eproline rich 22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e-6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e-5.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e-2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e3.3 Functional Enrichment Analysis of DEGs in Various Groups\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo gain deeper insights into the functions of DEGs between AKAV-infected and control cells, and to enhance our understanding of the role of host proteins in generating antiviral factors during AKAV infection, we conducted GO and KEGG enrichment analyses. GO enrichment analysis is a widely adopted approach for revealing the relationships between genes and functional terms. KEGG enrichment analysis, on the other hand, effectively illustrates the associations between genes and biological pathways. As depicted in Figure 3A and 3B, GO enrichment analysis of AKAV-infected BJSC and BTSC demonstrated significant enrichment in several biological processes (BPs). These included cytokine-mediated signaling pathwasy, specifically involving IL-1\u0026beta; and IL-1, and antiviral-related pathways such as the response to exogenous dsRNA, response to IFN-\u0026beta;, and regulation of the mitogen-activated protein kinase (MAPK) cascade. In contrast, in AKAV-infected MDOK cells, BP enrichment was primarily observed in the regulation of the MAPK cascade, protein localization to the plasma membrane, and extracellular matrix organization (Fig. 3C). KEGG enrichment analysis further indicated that DEGs were predominantly involved in pathways such as the TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, NOD-like receptor pathway, and herpes simplex virus 1 infection (Fig. 3D－F). The above results indicate that AKAV infection can induce the activation of inflammatory- and antiviral-associated pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4 Interferon and Inflammtory Responses are Activated in AKAV-infected Cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe demonstrated that AKAV infection induced substantial alterations in the transcriptomic profiles of host cells. To further validate these changes, we focused on interferon- and inflammation-associated cytokines. Using reverse transcription polymerase chain reaction (RT-PCR), we quantified the transcript levels of TNF-\u0026alpha;, IL-1\u0026beta;, CXCL8, CCL2, and Rnd1 in AKAV-infected cells. The fold changes in gene expression were calculated using the 2-\u0026Delta;\u0026Delta;Ct method. For normalization, we used the Ct values of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control for each sample. The results revealed that AKAV infection upregulated the expression of\u0026nbsp;TNF-\u0026alpha;, IL-1\u0026beta;, CXCL8, CCL2, and Rnd1\u0026nbsp;in all BTSC, BJSC, and MDOK cells (Fig. 4), which was in correspondence with the transcriptomic results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5 AKAV Induced Rnd1 Overexpression Through TNF-\u003c/em\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003cem\u003e\u0026nbsp;Signaling Pathway\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince Rnd1 expression was significantly upregulated in all AKAV-infected cells described above, we delved deeper into exploring its function and mechanism. MDOK cells were transfected with either the Rnd1-encoding plasmid (pCMV-Flag-Rnd1) or the vector backbone (pCMV-Flag-6C). After transfection, the cells were infected with AKAV. To determine the viral load, we employed two methods: the TCID\u003csub\u003e50\u003c/sub\u003e assay and WB. Analyses were carried out at 12 hpi and 24 hpi. As depicted in Fig. 5, after AKAV infection, transfection of MDOK cells with pCMV-Flag-Rnd1 resulted in a substantial reduction in AKAV viral load. This was evident in both the TCID\u003csub\u003e50\u003c/sub\u003e assay and WB analysis, when compared to control cells transfected with the vector backbone. To further investigate the role of TNF-\u0026alpha;\u0026nbsp;in AKAV-induced Rnd1 overexpression, we employed two experimental approaches. Ten ng/mL of recombinant TNF-\u0026alpha;\u0026nbsp;(purchased from MCE, China) was added to the cell culture, or TNF-\u0026alpha;-targeting siRNA (Table 1) was transfected into cells. Twelve hours later, cells were infected with AKAV at an MOI of 0.1, and the viral load was detected in 12 hpi and 24 hpi by TCID\u003csub\u003e50\u003c/sub\u003e and WB. As depicted in Fig. 5B, the addition of TNF-\u0026alpha; significantly upregulated Rnd1 expression. Concomitantly, there was a reduction in AKAV viral load. Conversely, when TNF-\u0026alpha; expression was silenced using siRNA, Rnd1 expression decreased. This was accompanied by an upregulation of AKAV replication (Fig. 5C). These results suggest that TNF-\u0026alpha; plays a crucial role in modulating Rnd1 expression and, subsequently, influences the replication of AKAV within host cells.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs one member of bunyavirus, AKAV is a Culicoides-borne virus and is teratogenic to the fetus of cattle and small ruminant species. When the cow or sheep was infected with AKAV during pregnancy, the adult cattle generally do not show clinical symptoms, while the fetus may develop with congenital malformations, including hydranencephaly, poliomyelitis and arthrogryposis [1]. Previous research has shown that AKAV can cross the placenta, and penetrate the blood-fetal barrier. This transplacental transmission leads to restricted reproductive disorders, resulting in congenital abnormalities [18]. However, the precise molecular mechanisms underpinning these processes remain unclear. Additionally, it is unknown whether AKAV can trigger reproductive disorders through alternative routes, such as via testicular cells. In this study, we systematically analyzed the infectivity of AKAV in multiple primary bovine cells. These included bovine turbinate cells, bovine duct epithelial cells, bovine lung epithelial cells, bovine mammary epithelial cells, BTSC, and BJSC. Our results demonstrate that AKAV could replicate efficiently in BTSC and BJSC. In contrast, only a small percentage (around 20%, data not shown) of bovine turbinate cells were infected. Through transcriptomic analysis, we found that AKAV infection significantly upregulates the expression of inflammatory-related genes, including TNF-α, IL-1β, CXCL8, and CCL2, which can partly explain why AKAV can cause reproductive disorders and joint related lesions[19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Rho family of GTPase consists of 20 members. In humans, these members are broadly classified into classic and atypical groups [20]. Classic Rho GTPases, prominently represented by RHOA, CDC42, and RAC1, function as molecular switches. They cycle between an inactive state, bound to DiPhosphate (GDP), and an active state, bound to Guanosine TriPhosphate (GTP) [21]. Given their significance in various cellular processes, classic Rho GTPase family members have been extensively studied. In contrast, atypical molecules such as the Rnd family proteins have received relatively less attention. Rnd1, also known as RHO6, is an atypical Rho GTPase. Rnd1 belongs to the RND subfamily, which also includes members Rnd2 (RHO7/RHON) and Rnd3 (RHO8/RHOE). All RND proteins feature the Rho-specific insert domain, a characteristic defining the Rho GTPase family. At the N-terminal region, Rnd1 and Rnd3 (but not Rnd2) possess a KERRA (Lys-Glu-Arg-Arg-Ala) sequence. This sequence facilitates their targeting to lipid rafts, thereby determining their localization at the plasma membrane [22]. Accumulating evidence from previous studies demonstrates that RNDs play crucial roles in axon guidance, cell cycle regulation, and tumorigenesis [23,24]. However, their relationship with virus infection remains largely unexplored. Previous study reported that Rnd1 can be induced by pro-inflammatory cytokines during viral and bacterial infections and provides protection against these pathogens through two distinct mechanisms [25]. First, Rnd1 acts as a regulator of intracellular calcium homeostasis, which inhibits RhoA activation to counter calcium fluctuations and block virus entry. Also, Rnd1 facilitates pro-inflammatory cytokines IL-6 and TNF-α through Plxnb1, which are highly effective against intracellular bacterial infections. However, in another study, TNF-α, IL-1β, and IL-6 also increased in rheumatoid synovial cells via Rnd1 overexpression [26,27]. In our study, we determined that following AKAV infection, the expression levels of Rnd1, IL-6, and TNF-α were significantly upregulated. TNF-α appears to play a crucial role in the overexpression of Rnd1. However, the precise molecular mechanisms underlying this regulatory relationship remain unclear and need further investigation.\u0026nbsp;\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, we utilized primary BTSC and BJSC to evaluate the infectivity of AKAV, confirming that both cell types are susceptible to AKAV TJ2016 strain. Through transcriptomic analysis, we found that AKAV infection significantly up-regulated interferon-stimulated genes, inflammation-related genes, and cytokines. Notably, we demonstrated that Rnd1 was significantly elevated in infected cells and showed that Rnd1 inhibits AKAV replication. Additionally, TNF-α was identified as a key inducer of Rnd1, providing mechanistic insights into antiviral regulation during AKAV infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDong-jie Chen: analysed data, drafted the manuscript and performed the laboratory experiments. Jing-jing Wang: analysed data and performed the laboratory experiments. Chao Sun: collected bovine primary cells. Fang Wei: collected data. Sheng-kui Xu: performed the laboratory experiments. Ru-yang Yu: analysed data. Shao-qiang Wu: supervised the study and finalized the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeclaration of competing interest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of this study declared that they do not have any conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2022YFD1802000), and the Natural Science Foundation of Beijing (6254044).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKurogi H, Inaba Y, Takahashi E, Sato K, Omori T, Miura Y, Goto Y, Fujiwara Y, Hatano Y, Kodama K, Fukuyama S, Sasaki N, Matumoto M (1976) Epizootic congenital arthrogryposis-hydranencephaly syndrome in cattle: isolation of Akabane virus from affected fetuses. 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Springer Semin Immunopathol 27(4):391\u0026ndash;408. https://doi.org/10.1007/s00281-006-0012-9\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Akabane virus, transcriptomic analysis, Rnd1, TNF-α signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-6646858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6646858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAkabane virus (AKAV), the etiological agent of Akabane disease, is known to induce congenital malformations and neuropathologies in the fetuses of pregnant cattle and sheep. To comprehensively analyze the protein alterations and precisely elucidate the molecular mechanism in AKAV-susceptible cells, two types of primary bovine cells, namely primary bovine testicular sertoli cells (BTSC) and primary bovine joint synovial cells (BJSC), were selected. These cells were confirmed to be effectively infected by the AKAV TJ2016 strain. Subsequently, RNA-sequencing technology was employed to further analyze the transcriptomic profiles of AKAV-infected BTSC, BJSC, and MDOK cells. The molecular features of AKAV-infected cells demonstrated a remarkable activation of antiviral signaling pathways. Notably, there was an upregulation in the expression levels of interferon-stimulated genes, as well as genes related to inflammation and cytokines. Through a comparison between infected and non-infected cells, it was revealed that IL-1β, TNF-α, CXCL8, CCL2 and Rnd1 were significantly up-regulated in AKAV-infected cells. Moreover, Rnd1 was found to inhibit the replication of AKAV and TNF-αplays an important role in the induction of Rnd1, which provides additional evidence for the regulation and function of Rnd1.\u003c/p\u003e","manuscriptTitle":"Transcriptomic Analysis Reveals Rnd1 as a Key Player in Antiviral Immunity Against Akabane virus via TNF-α pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 16:06:51","doi":"10.21203/rs.3.rs-6646858/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-13T05:27:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T05:19:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T13:35:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-05-27T08:00:48+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2025-05-16T03:34:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"adff81d0-1b48-43ed-b9d9-421235f13424","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T15:59:17+00:00","versionOfRecord":{"articleIdentity":"rs-6646858","link":"https://doi.org/10.1007/s00705-025-06477-1","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-11-28 15:56:56","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-06-17 16:06:51","video":"","vorDoi":"10.1007/s00705-025-06477-1","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06477-1","workflowStages":[]},"version":"v1","identity":"rs-6646858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6646858","identity":"rs-6646858","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}