Orf132: A Critical Gene for LSDV Replication and Its Role in ER Stress-Related Apoptosis

Orf132: A Critical Gene for LSDV Replication and Its Role in ER Stress-Related Apoptosis | 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 Orf132: A Critical Gene for LSDV Replication and Its Role in ER Stress-Related Apoptosis Yongtao Wang, Shiwei Zhang, Hailing Li, Yali Feng, Ying Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6261646/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 May, 2025 Read the published version in Virology Journal → Version 1 posted 11 You are reading this latest preprint version Abstract Lumpy skin disease (LSD), caused by lumpy skin disease virus (LSDV), is an emerging infectious disease in China that primarily affects cattle. LSDV and goatpox virus (GTPV) belong to the Capripoxvirus genus and exhibit high genomic homology, enabling cross-immunogenicity. Comparative genome analysis revealed that LSDV contains a unique gene, Orf132 , whose function remains uncharacterized. In this study, we first confirmed that the recombinant ORF132 protein exhibits immunoreactivity against sera from LSDV-infected cattle and GTPV-vaccinated cattle, and this cross-reactivity excluded the possibility of using the ORF132 protein to distinguish between LSDV and GTPV. To investigate the biological role of Orf132 , we generated an Orf132 deletion strain (LSDV-Δ Orf132 ). Compared with that of the wild-type LSDV, the replication capacity of LSDV-Δ Orf132 was reduced approximately tenfold, indicating that Orf132 is critical for viral replication. Transcriptomic analysis of infected MDBK cells revealed significant alterations in Endoplasmic reticulum(ER) protein processing and unfolded protein response(UPR) pathways following Orf132 deletion. RT-qPCR validation showed marked upregulation of ER stress markers including Grp78 , Chop , and Gadd34 . Subsequent apoptosis assays established that Orf132 deletion triggers CHOP-Caspase12-mediated apoptotic pathways. This dysregulated stress response cascade culminates in premature apoptotic scenarios, possibly resulting in a weakening of viral replication. Our findings collectively revealed that Orf132 is a critical gene for LSDV replication, plays an essential role in the virus's life cycle, and its deletion significantly impairs viral replication while inducing ER stress-related apoptosis. Lumpy skin disease virus Orf132 gene deletion ER stress reduced replication Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction LSD is a contagious bovine disease caused by LSDV and is classified as a notifiable disease by the World Organisation for Animal Health[ 1 ]. Clinical manifestations include skin nodules, emaciation, and reduced productivity, with mortality rates averaging 10% and infection rates ranging from 5–45% in endemic regions [ 2 , 3 ]. Since its first discovery in Zambia in 1929, LSD has rapidly spread across European and African countries[ 4 ]. On August 3, 2019, LSD was first reported in multiple provinces in China, including Jiangxi, Fujian, Anhui, Guangdong, Zhejiang, Xinjiang Uygur Autonomous Region, and Taiwan[ 5 ]. Vector control, restrictions on animal movement and extensive vaccination are effective strategies for controlling and eradicating LSD. The cross-immunogenicity between LSDV and GTPV attenuated vaccines underscores their shared antigenic properties. The LSDV genome ranges from 145–152 kbp in length and contains 156 potential open reading frames (ORFs)[ 6 ]. While homologous proteins of some LSDV genes are conserved across poxviruses, the functions of most genes have been inferred through their sequence similarity to known poxvirus proteins. However, LSDV harbors unique genes associated with host range and virulence, whose functions remain largely uncharacterized. The Capripoxvirus (CaPV) genus includes LSDV, sheeppox virus (SPPV), and GTPV, with nucleotide sequence homology exceeding 96% among these viruses[ 7 , 8 ]. In nature, LSDV primarily infects cattle, whereas GTPV and SPPV target goats and sheep [ 9 ]. These viruses diverged from a common ancestor through host range specialization [ 10 ]. Compared with LSDV, SPPV and GTPV lack nine genes implicated in virulence and host adaptation, including LSDV-specific Orf132 and several poxvirus homologs. These deleted genes include two copies each of myxomavirus M003.2 ( Orf 002) and M004.1 ( Orf 155), the IL-1 receptor ( Orf 013), and vaccinia virus orthologs F11L ( Orf 026), N2L ( Orf 009), and K7R ( Orf 136)[ 11 , 12 ]. For example, Orf013 encodes a protein homologous to VACV WR B15R , whose deletion may disrupt IL-1-mediated immune regulation and attenuate virulence [ 13 ]. Similarly, Orf136 shares sequence similarity with VACV A52R , which encodes an intracellular antagonist of the IL-1 and Toll-like receptor signaling pathways. The absence of these genes in SPPV/GTPV genome suggests their specialized role in bovine hosts[ 12 ]. Unique to LSDV, Orf132 encodes a protein with no detectable homology to other known viral or host proteins, and its functional significance remains unclear. Elucidating the role of this unique gene is critical for understanding LSDV-host interactions and poxvirus host specificity. In this study, we observed that the LSDV ORF132 protein exhibits strong immunogenicity but cross-reacts with GTPV sera. To investigate its function, we generated an LSDV-Δ Orf132 deletion strain via homologous recombination, in which Orf132 was replaced with EGFP. In vitro analysis revealed that Orf132 deletion reduced final titers by approximately 10-fold compared with those of wild-type LSDV. Furthermore, we determined that LSDV-Δ Orf132 infection induced ER stress-associated apoptosis in MDBK cells through the CHOP‒Caspase-12 pathway. These findings provide preliminary insights into the role of Orf132 in modulating virus replication and host cell apoptosis. Materials and Methods Plasmids, Protein, Cells, Virus and Serum The pET32a vector was maintained in our laboratory. The Orf132 gene was amplified and inserted into the pET32a vector (EcoRI/XhoI sites) to construct the pET32a- Orf132 plasmid. Competent E. coli BL21(DE3) cells were transformed with this plasmid and induced with 1 mM IPTG (Invitrogen, USA) for 3 hours. The ORF protein was purified via affinity chromatography. The transfer fragment (LSDV- Orf132 -L-EGFP-R) contained homologous arms flanking the Orf132 gene and the EGFP gene, which was driven by the VACV P7.5K promoter. A poxvirus transcription termination sequence was included downstream of each gene. MDBK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, CellMax, China) and penicillin-streptomycin (Solarbio, China). For LSDV infection experiments, the medium was replaced with DMEM containing 2% FBS. The LSDV strain, LSDV-positive cattle sera (LSDV+), and standard sera were provided by the China Institute of Veterinary Drug Control. GTPV-positive cattle sera (GTPV+) were maintained in our laboratory. All LSDV experiments were conducted in a biosafety level 3 facility approved by the Chinese Ministry of Agriculture. Antigenicity Determination of the ORF132 protein ELISAs were performed to analyze the reactivity of the ORF132 protein with LSDV + and GTPV + sera. ELISA plates (Beyotime, China) were coated with 50 µg of purified ORF132 protein overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween-20) and blocked with 5% skim milk in PBST for 2 hours. LSDV+, GTPV+, and LSDV standard sera were added to the wells, followed by incubation at room temperature for 2 hours. Bound antibodies were detected using rabbit anti-bovine IgG-HRP (SE233, Solarbio, China). Bioinformatic Analysis Linear B-cell epitopes in Orf132 were predicted via the Bepipred Linear Epitope Prediction 2.0 tool (IEDB Analysis Resource, http://tools.iedb.org/bcell/ ). BLASTp ( http://blast.ncbi.nlm.nih.gov ) identified cross-reactive sequences in GTPV (Taxid:186805) with ≥ 4 amino acid matches. Generation of Orf132 Gene Deletion Strain MDBK cells (70–80% confluency) were transfected with the LSDV- Orf132 -L-EGFP-R vector using Lipofectamine™ LTX (Invitrogen, USA). After 6 hours, cells were infected with LSDV to allow homologous recombination. Recombinant viruses were isolated by picking GFP-positive plaques and limited dilution. Deletion was confirmed via PCR with primers targeting the homologous arms. Viral Growth Curve MDBK cells (70–80% confluency) were infected with LSDV-Δ Orf132 or LSDV (MOI = 1). After 2 hours, the medium was replaced with DMEM containing 2% FBS. Virus samples (infected cells with supernatant) were collected daily for 5 days, freeze-thawed three times, and titrated.. RNA-seq Analysis LSDV-∆ Orf132- and LSDV-infected MDBK cells at an MOI of 1 at 24 hpi, with a blank control group set up simultaneously. Total RNA was then extracted from each sample via TRIzol reagent (Vazyme Biotech, Nanjing, China) for transcriptome sequencing. qPCR Validation of ER Stress-Related Genes On the basis of the transcriptome sequencing results, this study focused on cytokines involved in heat shock and ER stress responses regulated by the LSDV ORF132 protein. RNA was extracted, and the HiScript II One Step qRT‒PCR SYBR Green Kit (Vazyme Biotech, Nanjing, China) was used for fluorescence quantitative detection. Primers were designed to validate the relative mRNA levels of GRR78, CHOP, GAAD34, HSPA6, HSPA1A, DNAJB1, and GAPDH in the samples. With the GAPDH gene used as an internal reference, the relative expression levels were calculated via the 2 -△△CT formula. Validation that LSDV-∆ Orf132 induced ER stress-associated apoptosis. The cells were infected with LSDV- ∆Orf132 and LSDV at an MOI of 1 in 12-well cell culture plates, with a blank control set up. After 24 hours, the cell samples were washed three times with PBS. Each well was treated with 120 µl of RIPA lysis buffer (R0010, Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. The protein concentration was quantified via the BCA system (Beyotime, Shanghai, China), followed by the addition of 5× loading buffer and boiling at 95°C for 10 minutes. After SDS‒PAGE, the proteins were transferred to nitrocellulose (NC) membranes at a constant current of 300 mA. The membranes were blocked with skim milk at 37°C for 2 hours, incubated overnight at 4°C with primary antibodies (GRP78, A23453, Ablconal, WuHan, China; CHOP, A20987, Ablconal, WuHan, China; Caspase-12, A22864, Ablconal, WuHan, China; GAPDH, 60004, Proteintech, WuHan, China), and incubated with secondary antibodies (goat anti-rabbit IgG, A0208, Beyotime, Shanghai, China) at room temperature. Finally, the membranes were developed for protein bands in the developing machine, with thorough washing between each step via TBST buffer containing 0.5% Tween-20. To detect the degree of MDBK cell apoptosis induced by LSDV- ∆Orf132 and LSDV, a one-step TUNEL apoptosis detection kit (Beyotime, Shanghai, China) was used according to the manufacturer's instructions. When apoptosis occurs, the cells exhibit bright red fluorescence under a microscope. The cells were observed, and the fluorescence intensity was measured via GraphPad Prism software 6.01. Statistical Analysis The data are presented as the means ± standard deviations (SDs). Statistical analyses were performed via t tests with GraphPad Prism software. p values indicate significant differences (* p < 0. 05; ** p < 0. 01; *** p < 0. 001; and **** p < 0. 0001); ns indicates no significant difference. Results Orf132 Exhibits Immunogenicity but Cross-Reacts with GTPV Sera The ORF132 protein was expressed in E. coli BL21 (DE3) and confirmed via SDS‒PAGE. Proteins were purified by nickel affinity chromatography and and analyzed for antigenicity by Western blotting using LSDV-positive serum samples (Fig. 1 ). The recombinant ORF132 protein exhibited robust immunogenicity. Cross-reactivity analysis via indirect ELISA revealed that the ORF132 protein reacted with 24 out of 25 GTPV-positive serum samples (96% sensitivity, 1 indeterminate case) and showed 100% specificity against LSDV-negative sera (no false positives). Notably, all seven LSDV-positive samples reacted positively (100% positive rate), indicating that despite being LSDV specific, ORF132 cross-reacted with GTPV sera (Fig. 1 ). Conserved linear epitopes of ORF132 protein show high sequence identity with GTPV protein To elucidate the observed cross-reactivity between the ORF132 protein and GTPV-positive serum, BLASTp searches were conducted on the predicted peptide sequences to identify homologous sequences within GTPV proteins (Table 1 ). The search was performed using short input sequences of at least four consecutive amino acids to identify mimotopes in GTPV. The results revealed that all the predicted peptides were present in GTPV proteins, indicating a high level of sequence identity. Table 1 The sequence similarity between the predicted linear epitope peptide of ORF132 protein and GTPV protein was analyzed. Predicted peptides AA position Identity GTPV protein VSFNS 58–62 100.00% hypothetical protein GTPV _ gp097 100.00% ankyrin repeat protein 100.00% palmytilated EEV membrane glycoprotein 100.00% hypothetical protein GTPV _ gp081 75.00% DNA topoisomerase type I 75.00% hypothetical protein GTPV _ gp003 80.00% putative metalloprotease 100.00% putative viral membrane protein NDDV 72–75 100.00% hypothetical protein GTPV _ gp019 EVQIFSLDNS 88–97 43.75% DNA-binding virion core protein 71.43% CD47-like protein 80.00% hypothetical protein GTPV _ gp017 80.00% hypothetical protein GTPV _ gp108 100.00% hypothetical protein GTPV _ gp058 100.00% hypothetical protein GTPV _ gp083 100.00% hypothetical protein GTPV _ gp114 100.00% Hypothetical protein GTPV _ gp050 100.00% hypothetical protein GTPV _ gp128 57.14% N1R/p28-like protein 66.67% hypothetical protein GTPV _ gp142 80.00% hypothetical protein GTPV _ gp084 75.00% hypothetical protein GTPV _ gp129 66.67% hypothetical protein GTPV _ gp094 57.14% hypothetical protein GTPV _ gp109 80.00% hypothetical protein GTPV _ gp090 80.00% hypothetical protein GTPV _ gp071 80.00% phospholipase-D-like protein 54.55% late transcription factor VLTF-1 60.00% hypothetical protein GTPV _ gp095 57.14% RNA helicase NPH-II 100.00% G protein-coupled chemokine receptor-like protein 100.00% EIF2a-like PKR inhibitor NGIGNNVPTCIDGEVNNKKVKSE 121–143 85.71% RNA polymerase 48.00% double-strand RNA-binding protein 71.43% hypothetical protein GTPV _ gp044 66.67% hypothetical protein GTPV _ gp090 80.00% kelch-like protein 53.33% ankyrin repeat protein 55.56% hypothetical protein GTPV _ gp025 47.83% IL-18 binding protein 100.00% IL-1 receptor-like protein 100.00% Hypothetical protein GTPV _ gp047 83.33% late transcription factor VLTF-2 100.00% hypothetical protein GTPV _ gp052 38.89% late transcription factor VLTF-3 62.50% hypothetical protein GTPV _ gp132 66.67% ankyrin-like protein 100.00% EEV maturation protein 100.00% hypothetical protein GTPV _ gp067 83.33% hypothetical protein GTPV _ gp099 80.00% EEV Host range protein 100.00% hypothetical protein GTPV _ gp122 100.00% ankyrin-like protein 66.67% kelch-like protein 80.00% putative metalloprotease 50.00% RNA helicase NPH-II 100.00% EEV glycoprotein 100.00% hypothetical protein GTPV _ gp072 100.00% CD47-like protein 100.00% late transcription factor VLTF-1 60.00% hypothetical protein GTPV _ gp084 57.14% Hypothetical protein GTPV _ gp038 66.67% hypothetical protein GTPV _ gp083 80.00% hypothetical protein GTPV _ gp136 80.00% phospholipase-D-like protein 66.67% ankyrin repeat protein 80.00% hypothetical protein GTPV _ gp071 80.00% hypothetical protein GTPV _ gp017 75.00% poxvirus myristoylprotein 80.00% Ser/Thr kinase 42.11% hypothetical protein GTPV _ gp080 100.00% hypothetical protein GTPV _ gp005 100.00% hypothetical protein GTPV _ gp043 54.55% hypothetical protein GTPV _ gp127 80.00% hypothetical protein GTPV _ gp007 100.00% superoxide dismutase precursor 80.00% putative virion core protein 50.00% Hypothetical protein GTPV _ gp050 100.00% Hypothetical protein GTPV _ gp033 50.00% hypothetical protein GTPV _ gp040 46.67% hypothetical protein GTPV _ gp142 26.32% hypothetical protein GTPV _ gp029 100.00% DNA polymerase 60.00% putative DNA-binding virion core protein 50.00% hypothetical protein GTPV _ gp110 55.56% hypothetical protein GTPV _ gp108 60.00% ankyrin repeat protein 57.14% hypothetical protein GTPV _ gp128 66.67% hypothetical protein GTPV _ gp114 55.56% hypothetical protein GTPV _ gp079 100.00% IMV membrane protein 100.00% RNA polymerase subunit 100.00% Hypothetical protein GTPV _ gp032 100.00% poly(A) polymerase large subunit 100.00% kelch-like protein 66.67% hypothetical protein GTPV _ gp075 50.00% hypothetical protein GTPV _ gp111 100.00% hypothetical protein GTPV _ gp076 100.00% hypothetical protein GTPV _ gp003 80.00% hypothetical protein GTPV _ gp082 41.67% N1R/p28-like protein 100.00% hypothetical protein GTPV _ gp077 100.00% DNA-binding virion core protein 100.00% hypothetical protein GTPV _ gp006 100.00% hypothetical protein GTPV _ gp098 100.00% DNA topoisomerase type I 100.00% hypothetical protein GTPV _ gp129 29.41% hypothetical protein GTPV _ gp094 100.00% hypothetical protein GTPV _ gp097 100.00% hypothetical protein GTPV _ gp106 100.00% hypothetical protein GTPV _ gp129.5 100.00% sulfhydryl oxidase 100.00% hypothetical protein GTPV _ gp074 100.00% hypothetical protein GTPV _ gp001 100.00% hypothetical protein GTPV _ gp002 100.00% hypothetical protein GTPV _ gp034 100.00% DNA-binding phosphoprotein 100.00% hypothetical protein GTPV _ gp120 100.00% poly(A) polymerase small subunit 100.00% hypothetical protein GTPV _ gp130 100.00% hypothetical protein GTPV _ gp086 50.00% hypothetical protein GTPV _ gp103 QDSLDKE 145–151 100.00% IL-1 receptor-like protein 100.00% hypothetical protein GTPV _ gp089 80.00% hypothetical protein GTPV _ gp084 66.70% hypothetical protein GTPV _ gp067 80.00% ankyrin-like protein MWFKKH 168–173 100.00% double-strand RNA-binding protein 75.00% EEV Host range protein 100.00% hypothetical protein GTPV _ gp075 LSDV-Δ Orf 132 Displays Attenuated Replication Using homologous recombination technology, the gene deletion strain LSDV-Δ Orf132 was constructed (Fig. 2 ). Continuous purification was performed on MDBK using limited dilution method, and the genomes of LSDV and Δ Orf132 recombinant viruses were extracted. Specific primers(Table 2 ) for the homologous arms on both sides of the Orf132 gene were used for PCR amplification to evaluate the purity of the recombinant virus. After 9 generations of continuous purification, the single-gene deletion strain LSDV-Δ Orf132 was obtained (Fig. 2 ). To determine the effect of Orf132 deletion on LSDV replication, we measured the replication kinetics of LSDV-Δ Orf132 and LSDV (Fig. 2 ). The results showed that the Orf132 gene deletion affects viral replication, and the LSDV- ΔOrf13 2 gene deletion strain had significantly lower viral titers than the parental strain at most time points in vitro. These findings suggest that Orf132 gene enhances LSDV replication on MDBK and may affect LSDV adaptation to cattle. Table 2 Primers were used for constructing shuttle plasmids and detecting the purity of recombinant viruses Primers Sequence (5–3′) Description LSDV-132flank-L-F1 CCGAAAATGGGAGCACCATTTCC For left arm LSDV-132flank-L-R1 AAAAGCGGGTGGGTTTGGAATTAGTTTTATAATTTATATATTT LSDV-132-7.5K-F2 TTTTTAAATATATAAATTATAAAACTAATTCCAAACCCACCCGC For P7.5k promoter LSDV-132-7.5K-R2 CCCTTGCTCACCATTGATTGCTATTG LSDV-132-EGFP-F3 TCAATAGCAATCAATGGTGAGCAAGGGCGAG For maker gene LSDV-132-EGFP-R3 AAAAGTATAAGTATATTTTTTGTTTTACTTGTACAGCTCGTCCATGCCG LSDV-132flank-R-F4 TGGACGAGCTGTACAAGTAAAACAAAAAATATACTTATACT For Right arm LSDV-132flank-R-R4 CGAAGGGAACGCACTGGT LSDV- ∆Orf132 -F TTGATGGAAAAGATCCGTCTAT Detect the purity LSDV- ∆Orf132 -R TTACTTGTACAGCTCGTCC Transcriptomic Profiling Links Orf132 to ER Stress Regulation A total of 1,173 differentially expressed genes were identified in the LSDV and LSDV-Δ Orf132 infection groups and the blank cell control group (Supplemental Fig. 1). There were 444 DEGs common between the LSDV wild-type group and the LSDV-Δ Orf132 recombinant virus group, with 242 genes upregulated and 202 genes downregulated. GO enrichment analysis and KEGG pathway enrichment analysis were subsequently performed on the DEGs. The GO analysis results revealed that the DEGs were enriched mainly in processes such as negative regulation of cellular metabolic processes, negative regulation of biosynthetic processes, negative regulation of cellular macromolecule biosynthetic processes, and response to unfolded proteins(Supplemental Fig. 2). The KEGG analysis resultssuggest that deletion virus infection may activate the host immune response and interfere with host cytokine signaling, possibly affecting viral regulation of immune escape (Supplemental Fig. 3). The FoxO signaling pathway is involved in apoptosis, metabolism, and stress responses, and its activation may be associated with cell injury or metabolic reprogramming due to viral infection. Orf132 deletion may affect the ability of viruses to regulate immune escape. Notably, all 12 genes involved in the response to the unfolded protein pathway were strongly upregulated in the LSDV- ΔOrf132 -infected group, suggesting the activation of the URP. The expression of 10 genes associated with the ER stress pathway significantly differed during in vivo infection by LSDV-Δ Orf132 (Fig. 3 ). These analyses suggest that Orf132 may play a regulatory role in the host ER stress response. To further confirm the differential gene expression at 24 hours post infection of MDBK cells with LSDV and LSDV-Δ Orf132 , we selected three ER marker genes (Grp78, Chop, and Gadd34) and heat shock proteins (Hspa1a, Hsph1, and Hsp40) for RT‒qPCR validation from the RNA-seq data, Specific primers see(Table 3 ). Our results (Fig. 3 ) revealed that, at 24 hours post infection , the mRNA levels of Grp78, Chop, Gadd34, Hspa1a, Hsph1, Hsp40, and Dnajb1 were significantly elevated in MDBK cells infected with LSDV-ΔOrf132 compared with those in the blank control group. In contrast, after 24 h of infection of MDBK cells with LSDV, the mRNA levels of Grp78, Chop, Gadd34, Hspa1a, Hsph1, Hsp40, and Dnajb1 were not significantly different. The changes in the mRNA expression levels of these genes were consistent with the RNA-seq results and also suggested activation of cellular stress. Table 3 Primers were used to measure gene expression through real-time quantitative PCR Primers Sequence (5–3′) GRP78-F CCGAAAATGGGAGCACCATTTCC GRP78-R AAAAGCGGGTGGGTTTGGAATTAGTTTTATAATTTATATATTT Chop-F TTTTTAAATATATAAATTATAAAACTAATTCCAAACCCACCCGC Chop-R CCCTTGCTCACCATTGATTGCTATTG GAAD34-F TCAATAGCAATCAATGGTGAGCAAGGGCGAG GAAD34-R AAAAGTATAAGTATATTTTTTGTTTTACTTGTACAGCTCGTCCATGCCG HSPA1A-F TGGACGAGCTGTACAAGTAAAACAAAAAATATACTTATACT HSPA1A-R CGAAGGGAACGCACTGGT HSPH1-F TTGATGGAAAAGATCCGTCTAT HSPH1-R TTACTTGTACAGCTCGTCC DNAJB1-F TTGACCATCGAAGTGAAGCG DNAJB1-R ATCGGCTGGAATGTTGTTGG HSPA6-Q-F TAGAGATAAGATTCCTGAAGAG HSPA6-Q-R ATAGAGCCTGGAGAAGAT LSDV-Δ Orf 132 Activates ER Stress-Related Apoptosis in MDBK Cells The RNA-Seq and RT‒qPCR results revealed that the deletion of Orf132 affected the transcription of ER stress-related genes. To confirm the role of Orf132 deletion in ER stress, we infected MDBK cells with LSDV-Δ Orf132 and LSDV at an MOI of 1 and found that the expression of GRP78 and CHOP significantly increased at 24 hpi in the LSDV-Δ Orf132 infection group (Fig. 4 ). These findings indicate that LSDV-Δ Orf132 infection induces ER stress in MDBK cells. Both the full-length and the activated fragment forms of the ER stress-related apoptosis effector Caspase-12 significantly increased at 24 hpi. We subsequently used an in situ cell death detection kit for TUNEL staining to assess the levels of apoptosis induced by LSDV and LSDV-Δ Orf132 infection. At 24 hpi, the level of apoptosis in the LSDV-Δ Orf132 infection group was significantly greater than that in the LSDV infection group (Fig. 4 ). These results indicate that LSDV-Δ Orf132 infection mediates the initiation of ER stress-related apoptosis. Overall, the deletion of Orf132 induced an ER stress response in virus-infected cells and caused ER stress-related apoptosis by activating caspase-12. Discussion The ORF132 fusion protein was expressed in vitro and demonstrated good immunogenicity. Additionally, the protein expression of ORF132 during LSDV infection was verified. In vitro deletion of LSDV Orf132 led to replication defects. Using transcriptomic approaches, we revealed that Orf132 may influence ER stress-related pathways through host or viral mechanisms, ultimately leading to ER-associated apoptosis mediated by the CHOP‒Caspase-12 signaling pathway. There is high genomic homology between Capripoxviruses, with no significant differences in morphology, serologic features, or protein composition, making it difficult to distinguish LSDV from GTPV and SPPV via traditional serologic methods [ 14 , 15 ]. Orf132 is a gene of unknown function without known counterparts in the poxvirus family but is relatively conserved among different LSDV isolates. Currently, no research has investigated the role of Orf132 during LSDV infection. To investigate its function, we produced a recombinant ORF132 protein via E. coli BL21 (DE3) transformation.Western Blot analysis showed that the recombinant protein was recognized by LSDV-positive sera and exhibited strong immunogenicity. Notably, the protein exhibited 100% seroreactivity with LSDV-positive sera but showed significant cross-reactivity with 96% of GTPV-positive samples. This cross-reactivity, attributable to shared epitopes with GTPV-encoded proteins identified through BLASTp analysis, poses challenges for serological differentiation between LSDV and GTPV. Using LSDV-positive serum, we validated the immunogenicity of the LSDV ORF132 protein in vitro and found that the ORF132 recombinant protein can specifically react with positive bovine serum. These findings demonstrated that the LSDV ORF132 protein has good immunogenicity and confirmed the expression of the Orf132 gene during LSDV infection. However, the protein exhibited significant cross-reactivity with GTPV-positive sera, which hinders our ability to study it at the protein level in vitro. Bioinformatics analysis revealed that this may be due to shared antigenic epitopes between the numerous proteins encoded by GTPV and ORF132, resulting in cross-reactivity. LSDV has a large genome with many encoded genes. Therefore, constructing strains with specific single-gene deletions allows direct observation of the functions of unknown LSDV genes, which is beneficial for revealing their roles in the viral lifecycle. Through the construction of an LSDV Orf132 gene deletion strain, this study revealed for the first time the critical regulatory role of this gene in the viral replication cycle. In vitro infection experiments revealed that, compared with the parental strain, LSDV-Δ Orf132 significantly impaired replication in MDBK cells, with viral titers ​reduced by more than 10-fold. These findings provide direct experimental evidence for the essential contribution of Orf132 to viral fitness, highlighting its nonredundant function in sustaining optimal replication efficiency. Extensive research has shown that the induction of GRP78 is a hallmark of ER stress [ 16 , 17 ]. Upon ER stress activation, the ER-resident kinase PERK undergoes autophosphorylation, initiating phosphorylation of eukaryotic initiation factor 2α (eIF2α) [ 18 ]. Phosphorylation of eIF2α inhibits protein translation and synthesis, which is a crucial component of the UPR aimed at restoring ER homeostasis[ 19 ]. However, it also enhances the translation of activating transcription factor 4 (ATF4) [ 20 ]. ATF4 induces the expression of CHOP and GADD34. CHOP serves as a master regulator of ER stress-induced apoptosis, with its transcriptional upregulation signaling irreversible cellular stress [ 21 ], whereas GADD34 facilitates eIF2α dephosphorylation, establishing a negative feedback loop within the PERK pathway[ 22 ]. In this study, transcriptomic profiling revealed that Orf132 deletion induced differential expression of 444 host genes (242 upregulated and 202 downregulated), with pronounced dysregulation of the ER protein processing and UPR pathways. RT‒qPCR confirmed the significant upregulation of Grp78, Chop, and Gadd34 mRNA levels in Δ Orf13 2-infected cells, indicating premature activation of the PERK-eIF2α-ATF4 signaling axis. These findings demonstrate that Orf132 deficiency triggers aberrant ER stress responses, disrupting the balance between the adaptive UPR and apoptotic signaling. ER stress and the UPR are hallmark features of viral infections [ 23 , 24 ]. Viral replication often drives excessive synthesis of viral proteins, overwhelming the folding capacity of the ER and triggering ER stress through the accumulation of misfolded proteins. Prior studies have shown that LSDV infection induces ER stress in vitro and in vivo, which promotes apoptosis in bovine embryonic fibroblast (BEF) cells[ 25 ]. However, the contribution of individual viral proteins to the modulation of ER stress remains poorly characterized. While bulk viral protein synthesis can inherently induce ER stress, specific viral proteins may fine-tune this process by either suppressing or exacerbating stress signaling. Our findings indicate that the deletion of Orf132 leads to the early activation of ER stress at 24 hours post infection (hpi). Furthermore, the expression of the ER stress-associated apoptosis effector protein Caspase-12 significantly increased at 24 hpi, indicating activation of the UPR pathway. This leads to apoptosis induction through CHOP-mediated proapoptotic signaling and Caspase-12 activation. This dysregulated stress response cascade culminates in premature apoptotic scenarios, possibly resulting in a decrease in viral replication. Crucially, Orf132 functions as a negative regulator of ER stress, potentially through interactions with host or viral factors that delay UPR activation. ER stress-mediated apoptosis plays a pivotal role in viral replication mechanisms. Premature apoptosis may act as a host defense mechanism by restricting viral replication. For example, African swine fever virus (ASFV) suppresses CHOP transcription to prolong host cell survival and increase viral yields[ 26 ]. West Nile virus (WNV) achieves increased titers in CHOP-deficient cells resistant to ER stress-induced apoptosis[ 27 ]. Some viruses, such as hepatitis E virus (HEV), employ molecular chaperones such as Hsp70B, Hsp72, and Hsp40—induced by the ORF2 protein—to inhibit CHOP-mediated apoptosis[ 28 ]. In this study, orf132 , as a negative regulator of ER stress, prematurely activated the UPR pathway, leading to early activation of the CHOP-Caspase-12-mediated apoptosis pathway, which may limit viral replication. LSDV Orf132 may represent a component of a broader viral strategy for regulating ER stress, so systematic studies of other LSDV genes with potential roles in UPR regulation are warranted. Such investigations will aid in the rational development of attenuated vaccines for LSDV. Conclusion This study demonstrated that the ORF132 protein, while immunogenic, lacks specificity for LSDV serodiagnosis because of its cross-reactivity with the GTPV. Deletion of Orf132 attenuated viral replication and triggered ER stress-mediated apoptosis via CHOP-Caspase-12 activation. Premature apoptosis induction may disrupt viral release and dissemination of virus particles, thereby compromising LSDV replication. These findings suggest that Orf132 may modulate cell stress to optimize viral fitness. Abbreviations LSD Lumpy skin disease LSDV Lumpy skin disease virus GTPV goatpox virus ER endoplasmic reticulum ORFs open reading frames CaPV Capripoxvirus SPPV sheeppox virus ASFV African swine fever virus WNV West Nile virus HEV hepatitis E virus UPR unfolded protein response Hpi hours post infection eIF2α eukaryotic initiation factor 2α Declarations Consent for publication Not applicable Availability of data and materials The data used to support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was supported by the Liaoning Provincial Department of Education project(JYTYB2024068). Ethics approval and consent to participate Not applicable. Acknowledgments Not applicable Authors' contributions Yongtao Wang . , conceptualization, methodology, formal analysis, investigation, and writing-original draft; Shiwei Zhang . , formal analysis, data curation, and visualization; Hailing Li. , methodology and resources; Yali Feng . , investigation and resources; Ying Zhang . , project administration, conceptualization, supervision, and writing-review and editing. Authors' information Key Laboratory of Livestock Infectious Diseases, Ministry of Education, and Key Laboratory of Ruminant Infectious Disease Prevention and Control (East), Ministry of Agriculture and Rural Affairs, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, 120 Dongling Road, Shenyang 110866, China Yongtao Wang, Shiwei Zhang, Hailing Li, Yali Feng and Ying Zhang References Manual OTJBBO. Paris, France: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2012, Chap. 2.4. 3. 2012. Ahmed AM. Dessouki AAJIJoB, Biochemistry, Bioinformatics: Abattoir-based survey and histopathological findings of lumpy skin disease in cattle at Ismailia abattoir. 2013, 3(4):372. Tuppurainen ESM, Venter EH, Shisler JL, Gari G, Mekonnen GA, Juleff N, Lyons NA, De Clercq K, Upton C, Bowden TR, et al. Review: Capripoxvirus Diseases: Current Status and Opportunities for Control. Transbound Emerg Dis. 2017;64(3):729–45. Yadav P, Kumar A, Nath SS, Devasurmutt Y, Shashidhar G, Joshi M, Puvar A, Sharma S, Raval J, Pandit R, et al. Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks. BMC Genomics. 2024;25(1):196. Lu G, Xie J, Luo J, Shao R, Jia K, Li S. Lumpy skin disease outbreaks in China, since 3 August 2019. Transbound Emerg Dis. 2021;68(2):216–9. Tulman ER, Afonso CL, Lu Z, Zsak L, Kutish GF, Rock DL. Genome Lumpy Skin Disease Virus. 2001;75(15):7122–30. Black DN, Hammond JM, Kitching RP. Genomic relationship between capripoxviruses. Virus Res. 1986;5(2–3):277–92. Gershon PD, Black DN. A comparison of the genomes of capripoxvirus isolates of sheep, goats, and cattle. Virology. 1988;164(2):341–9. Berguido FJ, Gelaye E, Liu Y, Davaasuren B, Krstevski K, Djadjovski I, Ivanova E, Goujgoulova G, Loitsch A, Tuppurainen E et al. Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera. Microorganisms 2022, 10(10). Sprygin A, Mazloum A, van Schalkwyk A, Babiuk S. Capripoxviruses, leporipoxviruses, and orthopoxviruses: Occurrences of recombination. Front Microbiol. 2022;13:978829. Gershon PD, Black DN. A capripoxvirus pseudogene whose only intact homologs are in other poxvirus genomes. Virology. 1989;172(1):350–4. Tulman ER, Afonso CL, Lu Z, Zsak L, Sur JH, Sandybaev NT, Kerembekova UZ, Zaitsev VL, Kutish GF, Rock DL. The genomes of sheeppox and goatpox viruses. J Virol. 2002;76(12):6054–61. Spriggs MK, Hruby DE, Maliszewski CR, Pickup DJ, Sims JE, Buller RM, VanSlyke J. Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein. Cell. 1992;71(1):145–52. Babiuk S, Bowden TR, Boyle DB, Wallace DB, Kitching RP. Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle. Transbound Emerg Dis. 2008;55(7):263–72. Tuppurainen E, Dietze K, Wolff J, Bergmann H, Beltran-Alcrudo D, Fahrion A, Lamien CE, Busch F, Sauter-Louis C, Conraths FJ et al. Review: Vaccines and Vaccination against Lumpy Skin Disease. Vaccines 2021, 9(10). Liu H, Bowes RC 3rd, van de Water B, Sillence C, Nagelkerke JF, Stevens JL. Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2 + disturbances, and cell death in renal epithelial cells. J Biol Chem. 1997;272(35):21751–9. Rao RV, Peel A, Logvinova A, del Rio G, Hermel E, Yokota T, Goldsmith PC, Ellerby LM, Ellerby HM, Bredesen DE. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Lett. 2002;514(2–3):122–8. Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, Wek RC. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol. 1998;18(12):7499–509. Wang X, Xu L, Gillette TG, Jiang X, Wang ZV. The unfolded protein response in ischemic heart disease. J Mol Cell Cardiol. 2018;117:19–25. Vattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA. 2004;101(31):11269–74. Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11(4):381–9. Matsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, Nishimura K, Matsuo S. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology open. 2013;2(10):1084–90. Cirone M. ER Stress, UPR Activation and the Inflammatory Response to Viral Infection. Viruses 2021, 13(5). Gavilán E, Medina-Guzman R, Bahatyrevich-Kharitonik B, Ruano D. Protein Quality Control Systems and ER Stress as Key Players in SARS-CoV-2-Induced Neurodegeneration. Cells 2024, 13(2). Tan J, Liu Y, Li W, Zhang Y, Chen G, Fang Y, He X, Jing Z. Lumpy Skin Disease Virus Infection Activates Autophagy and Endoplasmic Reticulum Stress-Related Cell Apoptosis in Primary Bovine Embryonic Fibroblast Cells. Microorganisms 2023, 11(8). Galluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G. Viral control of mitochondrial apoptosis. PLoS Pathog. 2008;4(5):e1000018. Medigeshi GR, Lancaster AM, Hirsch AJ, Briese T, Lipkin WI, Defilippis V, Früh K, Mason PW, Nikolich-Zugich J, Nelson JA. West Nile virus infection activates the unfolded protein response, leading to CHOP induction and apoptosis. J Virol. 2007;81(20):10849–60. John L, Thomas S, Herchenröder O, Pützer BM, Schaefer S. Hepatitis E virus ORF2 protein activates the pro-apoptotic gene CHOP and anti-apoptotic heat shock proteins. PLoS ONE. 2011;6(9):e25378. Additional Declarations No competing interests reported. Supplementary Files supplement1.pdf Supplementaryfile2ThefulluncroppedGelsandBlotsimages.pdf Cite Share Download PDF Status: Published Journal Publication published 30 May, 2025 Read the published version in Virology Journal → Version 1 posted Editorial decision: Revision requested 13 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviews received at journal 05 Apr, 2025 Reviews received at journal 01 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 30 Mar, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Editor assigned by journal 20 Mar, 2025 Submission checks completed at journal 20 Mar, 2025 First submitted to journal 19 Mar, 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. 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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-6261646","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432766962,"identity":"4f35c7f7-72e4-4b9e-894c-8b5e70f44ac1","order_by":0,"name":"Yongtao Wang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yongtao","middleName":"","lastName":"Wang","suffix":""},{"id":432766964,"identity":"24c3b881-1d12-4800-8b37-5712076fae9c","order_by":1,"name":"Shiwei Zhang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shiwei","middleName":"","lastName":"Zhang","suffix":""},{"id":432766966,"identity":"ec32642e-69a2-4ed7-8086-af989b3f82a5","order_by":2,"name":"Hailing Li","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hailing","middleName":"","lastName":"Li","suffix":""},{"id":432766968,"identity":"72493065-2f92-4235-9295-4665b9fa7364","order_by":3,"name":"Yali Feng","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Feng","suffix":""},{"id":432766970,"identity":"5dcfe847-f7a7-4bf8-bbd1-a72a02d8f2e6","order_by":4,"name":"Ying Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDACZiBmbAAxmA8+gIoZEKmFnS0ZppSAFgaYFn4eMwmitBgc5z388ucOuzx5Z7a0yp9tdokN7M3bJBhq7uDWcpgvzULyTHKx4WHmY7d525ITG3iOlUkwHHuGRwuPmYFhG3Pixma2tNuMQEaDRI6ZBGPDYfxaEtvqgVp4zAp/AhkN8m8IajF+cLDtcOJ8Zh4zBl4go0GCB78WSaAtjI1txxM3MLMlS/OcO27cxpNWbJFwDLcWvvNnjD/+bKtOnN9/+ODHH2XVsv3shzfe+FCDW4vCAQY2cHQYHAASjGwMDGwgXgJODQwM8g0MzB+gDCD4g0fpKBgFo2AUjFgAAKlBVunmrxSqAAAAAElFTkSuQmCC","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-19 12:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6261646/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6261646/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-025-02813-8","type":"published","date":"2025-05-30T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79071512,"identity":"934ef3de-d680-4e9a-a82c-0b17af4b256f","added_by":"auto","created_at":"2025-03-24 06:12:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":186282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and purification of the recombinant protein ORF132.\u003c/strong\u003e (A) Induced expression of the recombinant protein ORF132. M: protein marker; 1: empty plasmid induction for 3 hours; 2: recombinant plasmid induction for 3 hours. (B) Results of the purification of the recombinant protein ORF132. M: protein marker; 1: purified recombinant protein. (C) Western blot identification results using the standard positive control serum of cattle infected with LSDV as the primary antibody. M: protein marker; 1: empty vector sample after induction; 2: purified recombinant protein. (D) The purified ORF132 protein was used to coat the ELISA plate, and the red column represents the bovine standard LSDV-positive serum sample. The green column represents the bovine standard LSDV-negative serum sample; the purple column represents the serum sample from cattle infected with LSDV; the orange column represents the bovine serum sample after inoculation with the GTPV attenuated live vaccine; the samples whose absorbance (450 nm) was greater than the dotted line (P/N \u0026gt; 2.1) were all positiveaccording to ELISA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/64b8185beb8ae27bcec3c86d.png"},{"id":79070799,"identity":"76ab2d7e-d6da-4c80-8f75-485fcd8487e8","added_by":"auto","created_at":"2025-03-24 06:04:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":638822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction strategy, purification and in vitro replication kinetics curve of LSDV-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e∆Orf132\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Schematic diagram of the \u003cstrong\u003eLSDV-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e∆Orf132\u003c/strong\u003e\u003c/em\u003e construction strategy. (B) Results of the purification of the Δ\u003cem\u003eOrf132\u003c/em\u003e gene deletion virus. (C) PCR was used to confirm the absence of wild-type LSDV. M: marker; 1: LSDV; 2: LSDV-\u003cem\u003e∆Orf132\u003c/em\u003e. (D) In vitro replication kinetic curves of LSDV and LSDV-\u003cem\u003e∆Orf132\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/24c861a048072fd196b13268.png"},{"id":79071514,"identity":"df3fe3b1-24b2-46aa-89c5-368d2a14b2b7","added_by":"auto","created_at":"2025-03-24 06:12:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing of the LSDV-infected group and the LSDV-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e∆Orf132\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-infected group and quantitative detection of the mRNA levels of ER stress marker genes. \u003c/strong\u003e(A) Heatmap showing the relative expression levels of genes involved in protein processing in the ER. (B) Heatmap showing the relative expression levels of genes involved in the response to the unfolded protein pathway. (C) Fluorescence quantitative detection of stress-related protein mRNA levels. (D) Quantitative detection of the mRNA levels of ER stress marker genes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/a220f323fd177bf8c33b4027.png"},{"id":79070803,"identity":"dc8a0f62-e6af-43fe-b670-d0baa9a521bd","added_by":"auto","created_at":"2025-03-24 06:04:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":243900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLSDV-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e∆Orf132\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection facilitates the activation of ER stress-related cell apoptosis in MDBK cells and increases the degree of apoptosis in MDBK cells.\u003c/strong\u003e MDBK cells were infected with LSDV (MOI = 1) for 24 h. Subsequently, proteins were harvested for Western blotting to detect ER stress-related cell apoptosis. (A) MDBK cells were infected or infected with LSDV (MOI = 1) for 24 h, after which the protein was collected. Compared with that in the LSDV infection group, ERstress-related apoptosis was activated. (B) MDBK cells were infected with LSDV and LSDV-\u003cem\u003e∆Orf132\u003c/em\u003e (MOI = 1) for 24 h and stained with an in situ cell death detection kit, with a blank control group used. (C) Relative expression was evaluated by detecting gray values. (D) Fluorescence intensity was measured to assess the level of apoptosis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/f8467ac2c89acc1030f375c3.png"},{"id":83782848,"identity":"ce6cfb02-40af-4038-b895-5d233f75ea0b","added_by":"auto","created_at":"2025-06-02 16:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4543065,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/1c12dea5-eb96-455f-8143-0b91b6defbac.pdf"},{"id":79071513,"identity":"8c6c5263-d7e8-4e3b-ab7a-ec17ba1653cc","added_by":"auto","created_at":"2025-03-24 06:12:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":408947,"visible":true,"origin":"","legend":"","description":"","filename":"supplement1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/9422261dbe5682259491ef78.pdf"},{"id":79070808,"identity":"ec2ff6a9-adfb-461e-8577-d63e8c44bfa8","added_by":"auto","created_at":"2025-03-24 06:04:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":386423,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2ThefulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6261646/v1/a38e369a3b796968499eeb69.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Orf132: A Critical Gene for LSDV Replication and Its Role in ER Stress-Related Apoptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLSD is a contagious bovine disease caused by LSDV and is classified as a notifiable disease by the World Organisation for Animal Health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Clinical manifestations include skin nodules, emaciation, and reduced productivity, with mortality rates averaging 10% and infection rates ranging from 5\u0026ndash;45% in endemic regions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since its first discovery in Zambia in 1929, LSD has rapidly spread across European and African countries[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On August 3, 2019, LSD was first reported in multiple provinces in China, including Jiangxi, Fujian, Anhui, Guangdong, Zhejiang, Xinjiang Uygur Autonomous Region, and Taiwan[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vector control, restrictions on animal movement and extensive vaccination are effective strategies for controlling and eradicating LSD. The cross-immunogenicity between LSDV and GTPV attenuated vaccines underscores their shared antigenic properties.\u003c/p\u003e \u003cp\u003eThe LSDV genome ranges from 145\u0026ndash;152 kbp in length and contains 156 potential open reading frames (ORFs)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While homologous proteins of some LSDV genes are conserved across poxviruses, the functions of most genes have been inferred through their sequence similarity to known poxvirus proteins. However, LSDV harbors unique genes associated with host range and virulence, whose functions remain largely uncharacterized. The Capripoxvirus (CaPV) genus includes LSDV, sheeppox virus (SPPV), and GTPV, with nucleotide sequence homology exceeding 96% among these viruses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In nature, LSDV primarily infects cattle, whereas GTPV and SPPV target goats and sheep [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These viruses diverged from a common ancestor through host range specialization [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Compared with LSDV, SPPV and GTPV lack nine genes implicated in virulence and host adaptation, including LSDV-specific \u003cem\u003eOrf132\u003c/em\u003e and several poxvirus homologs. These deleted genes include two copies each of myxomavirus M003.2 (\u003cem\u003eOrf\u003c/em\u003e002) and M004.1 (\u003cem\u003eOrf\u003c/em\u003e155), the IL-1 receptor (\u003cem\u003eOrf\u003c/em\u003e013), and vaccinia virus orthologs F11L (\u003cem\u003eOrf\u003c/em\u003e026), N2L (\u003cem\u003eOrf\u003c/em\u003e009), and K7R (\u003cem\u003eOrf\u003c/em\u003e136)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For example, \u003cem\u003eOrf013\u003c/em\u003e encodes a protein homologous to VACV WR \u003cem\u003eB15R\u003c/em\u003e, whose deletion may disrupt IL-1-mediated immune regulation and attenuate virulence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, \u003cem\u003eOrf136\u003c/em\u003e shares sequence similarity with VACV \u003cem\u003eA52R\u003c/em\u003e, which encodes an intracellular antagonist of the IL-1 and Toll-like receptor signaling pathways. The absence of these genes in SPPV/GTPV genome suggests their specialized role in bovine hosts[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Unique to LSDV, \u003cem\u003eOrf132\u003c/em\u003e encodes a protein with no detectable homology to other known viral or host proteins, and its functional significance remains unclear. Elucidating the role of this unique gene is critical for understanding LSDV-host interactions and poxvirus host specificity.\u003c/p\u003e \u003cp\u003eIn this study, we observed that the LSDV ORF132 protein exhibits strong immunogenicity but cross-reacts with GTPV sera. To investigate its function, we generated an LSDV-Δ\u003cem\u003eOrf132\u003c/em\u003e deletion strain via homologous recombination, in which \u003cem\u003eOrf132\u003c/em\u003e was replaced with EGFP. In vitro analysis revealed that \u003cem\u003eOrf132\u003c/em\u003e deletion reduced final titers by approximately 10-fold compared with those of wild-type LSDV. Furthermore, we determined that LSDV-Δ\u003cem\u003eOrf132\u003c/em\u003e infection induced ER stress-associated apoptosis in MDBK cells through the CHOP‒Caspase-12 pathway. These findings provide preliminary insights into the role of \u003cem\u003eOrf132\u003c/em\u003e in modulating virus replication and host cell apoptosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlasmids, Protein, Cells, Virus and Serum\u003c/h2\u003e\n \u003cp\u003eThe pET32a vector was maintained in our laboratory. The \u003cem\u003eOrf132\u003c/em\u003e gene was amplified and inserted into the pET32a vector (EcoRI/XhoI sites) to construct the pET32a-\u003cem\u003eOrf132\u003c/em\u003e plasmid. Competent \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells were transformed with this plasmid and induced with 1 mM IPTG (Invitrogen, USA) for 3 hours. The ORF protein was purified via affinity chromatography.\u003c/p\u003e\n \u003cp\u003eThe transfer fragment (LSDV-\u003cem\u003eOrf132\u003c/em\u003e-L-EGFP-R) contained homologous arms flanking the \u003cem\u003eOrf132\u003c/em\u003e gene and the EGFP gene, which was driven by the VACV P7.5K promoter. A poxvirus transcription termination sequence was included downstream of each gene.\u003c/p\u003e\n \u003cp\u003eMDBK cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, CellMax, China) and penicillin-streptomycin (Solarbio, China). For LSDV infection experiments, the medium was replaced with DMEM containing 2% FBS.\u003c/p\u003e\n \u003cp\u003eThe LSDV strain, LSDV-positive cattle sera (LSDV+), and standard sera were provided by the China Institute of Veterinary Drug Control. GTPV-positive cattle sera (GTPV+) were maintained in our laboratory. All LSDV experiments were conducted in a biosafety level 3 facility approved by the Chinese Ministry of Agriculture.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cstrong\u003eAntigenicity Determination of the ORF132 protein\u003c/strong\u003e\u003c/div\u003e\n\u003cp\u003eELISAs were performed to analyze the reactivity of the ORF132 protein with LSDV\u0026thinsp;+\u0026thinsp;and GTPV\u0026thinsp;+\u0026thinsp;sera. ELISA plates (Beyotime, China) were coated with 50 \u0026micro;g of purified ORF132 protein overnight at 4\u0026deg;C. The plates were washed three times with PBST (PBS\u0026thinsp;+\u0026thinsp;0.05% Tween-20) and blocked with 5% skim milk in PBST for 2 hours. LSDV+, GTPV+, and LSDV standard sera were added to the wells, followed by incubation at room temperature for 2 hours. Bound antibodies were detected using rabbit anti-bovine IgG-HRP (SE233, Solarbio, China).\u003c/p\u003e\n\u003ch3\u003eBioinformatic Analysis\u003c/h3\u003e\n\u003cp\u003eLinear B-cell epitopes in \u003cem\u003eOrf132\u003c/em\u003e were predicted via the Bepipred Linear Epitope Prediction 2.0 tool (IEDB Analysis Resource, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tools.iedb.org/bcell/\u003c/span\u003e\u003c/span\u003e). BLASTp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e) identified cross-reactive sequences in GTPV (Taxid:186805) with \u0026ge;\u0026thinsp;4 amino acid matches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of\u003c/strong\u003e \u003cstrong\u003eOrf132\u003c/strong\u003e \u003cstrong\u003eGene Deletion Strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDBK cells (70\u0026ndash;80% confluency) were transfected with the LSDV-\u003cem\u003eOrf132\u003c/em\u003e-L-EGFP-R vector using Lipofectamine\u0026trade; LTX (Invitrogen, USA). After 6 hours, cells were infected with LSDV to allow homologous recombination. Recombinant viruses were isolated by picking GFP-positive plaques and limited dilution. Deletion was confirmed via PCR with primers targeting the homologous arms.\u003c/p\u003e\n\u003ch3\u003eViral Growth Curve\u003c/h3\u003e\n\u003cp\u003eMDBK cells (70\u0026ndash;80% confluency) were infected with LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e or LSDV (MOI\u0026thinsp;=\u0026thinsp;1). After 2 hours, the medium was replaced with DMEM containing 2% FBS. Virus samples (infected cells with supernatant) were collected daily for 5 days, freeze-thawed three times, and titrated..\u003c/p\u003e\n\u003ch3\u003eRNA-seq Analysis\u003c/h3\u003e\n\u003cp\u003eLSDV-∆\u003cem\u003eOrf132-\u003c/em\u003e and LSDV-infected MDBK cells at an MOI of 1 at 24 hpi, with a blank control group set up simultaneously. Total RNA was then extracted from each sample via TRIzol reagent (Vazyme Biotech, Nanjing, China) for transcriptome sequencing.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eqPCR Validation of ER Stress-Related Genes\u003c/h2\u003e\n \u003cp\u003eOn the basis of the transcriptome sequencing results, this study focused on cytokines involved in heat shock and ER stress responses regulated by the LSDV ORF132 protein. RNA was extracted, and the HiScript II One Step qRT‒PCR SYBR Green Kit (Vazyme Biotech, Nanjing, China) was used for fluorescence quantitative detection. Primers were designed to validate the relative mRNA levels of GRR78, CHOP, GAAD34, HSPA6, HSPA1A, DNAJB1, and GAPDH in the samples. With the GAPDH gene used as an internal reference, the relative expression levels were calculated via the 2\u003csup\u003e-△△CT\u003c/sup\u003e formula.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eValidation that LSDV-∆\u003c/strong\u003e \u003cstrong\u003eOrf132\u003c/strong\u003e \u003cstrong\u003einduced ER stress-associated apoptosis.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe cells were infected with LSDV-\u003cem\u003e∆Orf132\u003c/em\u003e and LSDV at an MOI of 1 in 12-well cell culture plates, with a blank control set up. After 24 hours, the cell samples were washed three times with PBS. Each well was treated with 120 \u0026micro;l of RIPA lysis buffer (R0010, Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. The protein concentration was quantified via the BCA system (Beyotime, Shanghai, China), followed by the addition of 5\u0026times; loading buffer and boiling at 95\u0026deg;C for 10 minutes. After SDS‒PAGE, the proteins were transferred to nitrocellulose (NC) membranes at a constant current of 300 mA. The membranes were blocked with skim milk at 37\u0026deg;C for 2 hours, incubated overnight at 4\u0026deg;C with primary antibodies (GRP78, A23453, Ablconal, WuHan, China; CHOP, A20987, Ablconal, WuHan, China; Caspase-12, A22864, Ablconal, WuHan, China; GAPDH, 60004, Proteintech, WuHan, China), and incubated with secondary antibodies (goat anti-rabbit IgG, A0208, Beyotime, Shanghai, China) at room temperature. Finally, the membranes were developed for protein bands in the developing machine, with thorough washing between each step via TBST buffer containing 0.5% Tween-20.\u003c/p\u003e\n \u003cp\u003eTo detect the degree of MDBK cell apoptosis induced by LSDV-\u003cem\u003e∆Orf132\u003c/em\u003e and LSDV, a one-step TUNEL apoptosis detection kit (Beyotime, Shanghai, China) was used according to the manufacturer\u0026apos;s instructions. When apoptosis occurs, the cells exhibit bright red fluorescence under a microscope. The cells were observed, and the fluorescence intensity was measured via GraphPad Prism software 6.01.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eThe data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs). Statistical analyses were performed via t tests with GraphPad Prism software. p values indicate significant differences (* p\u0026thinsp;\u0026lt;\u0026thinsp;0. 05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0. 01; *** p\u0026thinsp;\u0026lt;\u0026thinsp;0. 001; and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0. 0001); ns indicates no significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOrf132\u003c/strong\u003e \u003cstrong\u003eExhibits Immunogenicity but Cross-Reacts with GTPV Sera\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ORF132 protein was expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) and confirmed via SDS‒PAGE. Proteins were purified by nickel affinity chromatography and and analyzed for antigenicity by Western blotting using LSDV-positive serum samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The recombinant ORF132 protein exhibited robust immunogenicity. Cross-reactivity analysis via indirect ELISA revealed that the ORF132 protein reacted with 24 out of 25 GTPV-positive serum samples (96% sensitivity, 1 indeterminate case) and showed 100% specificity against LSDV-negative sera (no false positives). Notably, all seven LSDV-positive samples reacted positively (100% positive rate), indicating that despite being LSDV specific, ORF132 cross-reacted with GTPV sera (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eConserved linear epitopes of ORF132 protein show high sequence identity with GTPV protein\u003c/h2\u003e\n\u003cp\u003eTo elucidate the observed cross-reactivity between the ORF132 protein and GTPV-positive serum, BLASTp searches were conducted on the predicted peptide sequences to identify homologous sequences within GTPV proteins (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The search was performed using short input sequences of at least four consecutive amino acids to identify mimotopes in GTPV. The results revealed that all the predicted peptides were present in GTPV proteins, indicating a high level of sequence identity.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe sequence similarity between the predicted linear epitope peptide of ORF132 protein and GTPV protein was analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePredicted peptides\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAA position\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIdentity\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGTPV protein\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"8\" align=\"left\"\u003e\n\u003cp\u003eVSFNS\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"8\" align=\"left\"\u003e\n\u003cp\u003e58\u0026ndash;62\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e100.00%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ehypothetical protein GTPV _ gp097\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e100.00%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eankyrin repeat protein\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003epalmytilated EEV membrane glycoprotein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp081\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e75.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA topoisomerase type I\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e75.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp003\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eputative metalloprotease\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eputative viral membrane protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eNDDV\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e72\u0026ndash;75\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp019\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"23\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEVQIFSLDNS\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"23\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e88\u0026ndash;97\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e43.75%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA-binding virion core protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e71.43%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCD47-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp017\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp108\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp058\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp083\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp114\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp050\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp128\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e57.14%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eN1R/p28-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp142\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp084\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e75.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp129\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp094\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e57.14%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp109\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp090\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp071\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ephospholipase-D-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e54.55%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003elate transcription factor VLTF-1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e60.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp095\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e57.14%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA helicase NPH-II\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eG protein-coupled chemokine receptor-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEIF2a-like PKR inhibitor\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"64\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eNGIGNNVPTCIDGEVNNKKVKSE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"64\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e121\u0026ndash;143\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e85.71%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA polymerase\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e48.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003edouble-strand RNA-binding protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e71.43%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp044\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp090\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ekelch-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e53.33%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin repeat protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e55.56%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp025\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e47.83%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-18 binding protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-1 receptor-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp047\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e83.33%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003elate transcription factor VLTF-2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp052\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e38.89%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003elate transcription factor VLTF-3\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e62.50%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp132\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEEV maturation protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp067\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e83.33%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp099\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEEV Host range protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp122\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ekelch-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eputative metalloprotease\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA helicase NPH-II\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEEV glycoprotein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp072\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCD47-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003elate transcription factor VLTF-1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e60.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp084\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e57.14%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp038\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp083\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp136\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ephospholipase-D-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin repeat protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp071\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp017\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e75.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003epoxvirus myristoylprotein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSer/Thr kinase\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e42.11%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp080\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp005\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp043\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e54.55%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp127\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp007\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003esuperoxide dismutase precursor\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eputative virion core protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp050\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp033\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp040\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e46.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp142\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e26.32%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp029\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA polymerase\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e60.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eputative DNA-binding virion core protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp110\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e55.56%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp108\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e60.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin repeat protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e57.14%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp128\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp114\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e55.56%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp079\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eIMV membrane protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA polymerase subunit\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHypothetical protein GTPV _ gp032\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003epoly(A) polymerase large subunit\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ekelch-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp075\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"26\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"26\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp111\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp076\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp003\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp082\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e41.67%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eN1R/p28-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp077\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA-binding virion core protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp006\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp098\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA topoisomerase type I\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp129\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e29.41%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp094\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp097\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp106\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp129.5\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003esulfhydryl oxidase\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp074\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp001\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp002\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp034\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA-binding phosphoprotein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp120\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003epoly(A) polymerase small subunit\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp130\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp086\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e50.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp103\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eQDSLDKE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e145\u0026ndash;151\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-1 receptor-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp089\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp084\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e66.70%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp067\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e80.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eankyrin-like protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMWFKKH\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e168\u0026ndash;173\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003edouble-strand RNA-binding protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e75.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEEV Host range protein\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e100.00%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehypothetical protein GTPV _ gp075\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eLSDV-\u0026Delta;\u003c/strong\u003e \u003cstrong\u003eOrf\u003c/strong\u003e \u003cstrong\u003e132 Displays Attenuated Replication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing homologous recombination technology, the gene deletion strain LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e was constructed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Continuous purification was performed on MDBK using limited dilution method, and the genomes of LSDV and \u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e recombinant viruses were extracted. Specific primers(Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) for the homologous arms on both sides of the \u003cem\u003eOrf132\u003c/em\u003e gene were used for PCR amplification to evaluate the purity of the recombinant virus. After 9 generations of continuous purification, the single-gene deletion strain LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e was obtained (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). To determine the effect of \u003cem\u003eOrf132\u003c/em\u003e deletion on LSDV replication, we measured the replication kinetics of LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e and LSDV (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The results showed that the \u003cem\u003eOrf132\u003c/em\u003e gene deletion affects viral replication, and the LSDV-\u003cem\u003e\u0026Delta;Orf13\u003c/em\u003e2 gene deletion strain had significantly lower viral titers than the parental strain at most time points in vitro. These findings suggest that \u003cem\u003eOrf132\u003c/em\u003e gene enhances LSDV replication on MDBK and may affect LSDV adaptation to cattle.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePrimers were used for constructing shuttle plasmids and detecting the purity of recombinant viruses\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimers\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSequence (5\u0026ndash;3\u0026prime;)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDescription\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\"\u003e\n\u003cp\u003eLSDV-132flank-L-F1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCGAAAATGGGAGCACCATTTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFor left arm\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132flank-L-R1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAAAAGCGGGTGGGTTTGGAATTAGTTTTATAATTTATATATTT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132-7.5K-F2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTTTTAAATATATAAATTATAAAACTAATTCCAAACCCACCCGC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFor P7.5k promoter\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132-7.5K-R2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCCTTGCTCACCATTGATTGCTATTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132-EGFP-F3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTCAATAGCAATCAATGGTGAGCAAGGGCGAG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFor maker gene\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132-EGFP-R3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAAAAGTATAAGTATATTTTTTGTTTTACTTGTACAGCTCGTCCATGCCG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132flank-R-F4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTGGACGAGCTGTACAAGTAAAACAAAAAATATACTTATACT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFor Right arm\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-132flank-R-R4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCGAAGGGAACGCACTGGT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-\u003cem\u003e∆Orf132\u003c/em\u003e-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTGATGGAAAAGATCCGTCTAT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDetect the purity\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLSDV-\u003cem\u003e∆Orf132\u003c/em\u003e-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTACTTGTACAGCTCGTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic Profiling Links\u003c/strong\u003e \u003cstrong\u003eOrf132\u003c/strong\u003e \u003cstrong\u003eto ER Stress Regulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1,173 differentially expressed genes were identified in the LSDV and LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection groups and the blank cell control group (Supplemental Fig.\u0026nbsp;1). There were 444 DEGs common between the LSDV wild-type group and the LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e recombinant virus group, with 242 genes upregulated and 202 genes downregulated. GO enrichment analysis and KEGG pathway enrichment analysis were subsequently performed on the DEGs. The GO analysis results revealed that the DEGs were enriched mainly in processes such as negative regulation of cellular metabolic processes, negative regulation of biosynthetic processes, negative regulation of cellular macromolecule biosynthetic processes, and response to unfolded proteins(Supplemental Fig.\u0026nbsp;2). The KEGG analysis resultssuggest that deletion virus infection may activate the host immune response and interfere with host cytokine signaling, possibly affecting viral regulation of immune escape (Supplemental Fig.\u0026nbsp;3). The FoxO signaling pathway is involved in apoptosis, metabolism, and stress responses, and its activation may be associated with cell injury or metabolic reprogramming due to viral infection.\u003cem\u003eOrf132\u003c/em\u003e deletion may affect the ability of viruses to regulate immune escape. Notably, all 12 genes involved in the response to the unfolded protein pathway were strongly upregulated in the LSDV-\u003cem\u003e\u0026Delta;Orf132\u003c/em\u003e-infected group, suggesting the activation of the URP. The expression of 10 genes associated with the ER stress pathway significantly differed during in vivo infection by LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These analyses suggest that \u003cem\u003eOrf132\u003c/em\u003e may play a regulatory role in the host ER stress response.\u003c/p\u003e\n\u003cp\u003eTo further confirm the differential gene expression at 24 hours post infection of MDBK cells with LSDV and LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e, we selected three ER marker genes (Grp78, Chop, and Gadd34) and heat shock proteins (Hspa1a, Hsph1, and Hsp40) for RT‒qPCR validation from the RNA-seq data, Specific primers see(Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Our results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed that, at 24 hours post \u003cem\u003einfection\u003c/em\u003e, the mRNA levels of Grp78, Chop, Gadd34, Hspa1a, Hsph1, Hsp40, and Dnajb1 were significantly elevated in MDBK cells infected with LSDV-\u0026Delta;Orf132 compared with those in the blank control group. In contrast, after 24 h of infection of MDBK cells with LSDV, the mRNA levels of Grp78, Chop, Gadd34, Hspa1a, Hsph1, Hsp40, and Dnajb1 were not significantly different. The changes in the mRNA expression levels of these genes were consistent with the RNA-seq results and also suggested activation of cellular stress.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePrimers were used to measure gene expression through real-time quantitative PCR\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimers\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSequence (5\u0026ndash;3\u0026prime;)\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\"\u003e\n\u003cp\u003eGRP78-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCGAAAATGGGAGCACCATTTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGRP78-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAAAAGCGGGTGGGTTTGGAATTAGTTTTATAATTTATATATTT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChop-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTTTTAAATATATAAATTATAAAACTAATTCCAAACCCACCCGC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChop-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCCTTGCTCACCATTGATTGCTATTG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGAAD34-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTCAATAGCAATCAATGGTGAGCAAGGGCGAG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGAAD34-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAAAAGTATAAGTATATTTTTTGTTTTACTTGTACAGCTCGTCCATGCCG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPA1A-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTGGACGAGCTGTACAAGTAAAACAAAAAATATACTTATACT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPA1A-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCGAAGGGAACGCACTGGT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPH1-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTGATGGAAAAGATCCGTCTAT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPH1-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTACTTGTACAGCTCGTCC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNAJB1-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTTGACCATCGAAGTGAAGCG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNAJB1-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eATCGGCTGGAATGTTGTTGG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPA6-Q-F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTAGAGATAAGATTCCTGAAGAG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHSPA6-Q-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eATAGAGCCTGGAGAAGAT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eLSDV-\u0026Delta;\u003c/strong\u003e \u003cstrong\u003eOrf\u003c/strong\u003e \u003cstrong\u003e132 Activates ER Stress-Related Apoptosis in MDBK Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq and RT‒qPCR results revealed that the deletion of \u003cem\u003eOrf132\u003c/em\u003e affected the transcription of ER stress-related genes. To confirm the role of \u003cem\u003eOrf132\u003c/em\u003e deletion in ER stress, we infected MDBK cells with LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e and LSDV at an MOI of 1 and found that the expression of GRP78 and CHOP significantly increased at 24 hpi in the LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings indicate that LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection induces ER stress in MDBK cells. Both the full-length and the activated fragment forms of the ER stress-related apoptosis effector Caspase-12 significantly increased at 24 hpi. We subsequently used an in situ cell death detection kit for TUNEL staining to assess the levels of apoptosis induced by LSDV and LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection. At 24 hpi, the level of apoptosis in the LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection group was significantly greater than that in the LSDV infection group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicate that LSDV-\u0026Delta;\u003cem\u003eOrf132\u003c/em\u003e infection mediates the initiation of ER stress-related apoptosis. Overall, the deletion of \u003cem\u003eOrf132\u003c/em\u003e induced an ER stress response in virus-infected cells and caused ER stress-related apoptosis by activating caspase-12.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ORF132 fusion protein was expressed in vitro and demonstrated good immunogenicity. Additionally, the protein expression of ORF132 during LSDV infection was verified. In vitro deletion of LSDV \u003cem\u003eOrf132\u003c/em\u003e led to replication defects. Using transcriptomic approaches, we revealed that \u003cem\u003eOrf132\u003c/em\u003e may influence ER stress-related pathways through host or viral mechanisms, ultimately leading to ER-associated apoptosis mediated by the CHOP‒Caspase-12 signaling pathway.\u003c/p\u003e \u003cp\u003eThere is high genomic homology between Capripoxviruses, with no significant differences in morphology, serologic features, or protein composition, making it difficult to distinguish LSDV from GTPV and SPPV via traditional serologic methods [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eOrf132\u003c/em\u003e is a gene of unknown function without known counterparts in the poxvirus family but is relatively conserved among different LSDV isolates. Currently, no research has investigated the role of \u003cem\u003eOrf132\u003c/em\u003e during LSDV infection. To investigate its function, we produced a recombinant ORF132 protein via \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) transformation.Western Blot analysis showed that the recombinant protein was recognized by LSDV-positive sera and exhibited strong immunogenicity. Notably, the protein exhibited 100% seroreactivity with LSDV-positive sera but showed significant cross-reactivity with 96% of GTPV-positive samples. This cross-reactivity, attributable to shared epitopes with GTPV-encoded proteins identified through BLASTp analysis, poses challenges for serological differentiation between LSDV and GTPV. Using LSDV-positive serum, we validated the immunogenicity of the LSDV ORF132 protein in vitro and found that the ORF132 recombinant protein can specifically react with positive bovine serum. These findings demonstrated that the LSDV ORF132 protein has good immunogenicity and confirmed the expression of the \u003cem\u003eOrf132\u003c/em\u003e gene during LSDV infection. However, the protein exhibited significant cross-reactivity with GTPV-positive sera, which hinders our ability to study it at the protein level in vitro. Bioinformatics analysis revealed that this may be due to shared antigenic epitopes between the numerous proteins encoded by GTPV and ORF132, resulting in cross-reactivity.\u003c/p\u003e \u003cp\u003eLSDV has a large genome with many encoded genes. Therefore, constructing strains with specific single-gene deletions allows direct observation of the functions of unknown LSDV genes, which is beneficial for revealing their roles in the viral lifecycle. Through the construction of an LSDV \u003cem\u003eOrf132\u003c/em\u003e gene deletion strain, this study revealed for the first time the critical regulatory role of this gene in the viral replication cycle. In vitro infection experiments revealed that, compared with the parental strain, LSDV-Δ\u003cem\u003eOrf132\u003c/em\u003e significantly impaired replication in MDBK cells, with viral titers ​reduced by more than 10-fold. These findings provide direct experimental evidence for the essential contribution of \u003cem\u003eOrf132\u003c/em\u003e to viral fitness, highlighting its nonredundant function in sustaining optimal replication efficiency.\u003c/p\u003e \u003cp\u003eExtensive research has shown that the induction of GRP78 is a hallmark of ER stress [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Upon ER stress activation, the ER-resident kinase PERK undergoes autophosphorylation, initiating phosphorylation of eukaryotic initiation factor 2α (eIF2α) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Phosphorylation of eIF2α inhibits protein translation and synthesis, which is a crucial component of the UPR aimed at restoring ER homeostasis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, it also enhances the translation of activating transcription factor 4 (ATF4) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. ATF4 induces the expression of CHOP and GADD34. CHOP serves as a master regulator of ER stress-induced apoptosis, with its transcriptional upregulation signaling irreversible cellular stress [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], whereas GADD34 facilitates eIF2α dephosphorylation, establishing a negative feedback loop within the PERK pathway[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, transcriptomic profiling revealed that \u003cem\u003eOrf132\u003c/em\u003e deletion induced differential expression of 444 host genes (242 upregulated and 202 downregulated), with pronounced dysregulation of the ER protein processing and UPR pathways. RT‒qPCR confirmed the significant upregulation of Grp78, Chop, and Gadd34 mRNA levels in Δ\u003cem\u003eOrf13\u003c/em\u003e2-infected cells, indicating premature activation of the PERK-eIF2α-ATF4 signaling axis. These findings demonstrate that \u003cem\u003eOrf132\u003c/em\u003e deficiency triggers aberrant ER stress responses, disrupting the balance between the adaptive UPR and apoptotic signaling.\u003c/p\u003e \u003cp\u003eER stress and the UPR are hallmark features of viral infections [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Viral replication often drives excessive synthesis of viral proteins, overwhelming the folding capacity of the ER and triggering ER stress through the accumulation of misfolded proteins. Prior studies have shown that LSDV infection induces ER stress in vitro and in vivo, which promotes apoptosis in bovine embryonic fibroblast (BEF) cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the contribution of individual viral proteins to the modulation of ER stress remains poorly characterized. While bulk viral protein synthesis can inherently induce ER stress, specific viral proteins may fine-tune this process by either suppressing or exacerbating stress signaling. Our findings indicate that the deletion of \u003cem\u003eOrf132\u003c/em\u003e leads to the early activation of ER stress at 24 hours post infection (hpi). Furthermore, the expression of the ER stress-associated apoptosis effector protein Caspase-12 significantly increased at 24 hpi, indicating activation of the UPR pathway. This leads to apoptosis induction through CHOP-mediated proapoptotic signaling and Caspase-12 activation. This dysregulated stress response cascade culminates in premature apoptotic scenarios, possibly resulting in a decrease in viral replication. Crucially, \u003cem\u003eOrf132\u003c/em\u003e functions as a negative regulator of ER stress, potentially through interactions with host or viral factors that delay UPR activation. ER stress-mediated apoptosis plays a pivotal role in viral replication mechanisms. Premature apoptosis may act as a host defense mechanism by restricting viral replication. For example, African swine fever virus (ASFV) suppresses CHOP transcription to prolong host cell survival and increase viral yields[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. West Nile virus (WNV) achieves increased titers in CHOP-deficient cells resistant to ER stress-induced apoptosis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Some viruses, such as hepatitis E virus (HEV), employ molecular chaperones such as Hsp70B, Hsp72, and Hsp40\u0026mdash;induced by the ORF2 protein\u0026mdash;to inhibit CHOP-mediated apoptosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, \u003cem\u003eorf132\u003c/em\u003e, as a negative regulator of ER stress, prematurely activated the UPR pathway, leading to early activation of the CHOP-Caspase-12-mediated apoptosis pathway, which may limit viral replication. LSDV \u003cem\u003eOrf132\u003c/em\u003e may represent a component of a broader viral strategy for regulating ER stress, so systematic studies of other LSDV genes with potential roles in UPR regulation are warranted. Such investigations will aid in the rational development of attenuated vaccines for LSDV.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that the ORF132 protein, while immunogenic, lacks specificity for LSDV serodiagnosis because of its cross-reactivity with the GTPV. Deletion of \u003cem\u003eOrf132\u003c/em\u003e attenuated viral replication and triggered ER stress-mediated apoptosis via CHOP-Caspase-12 activation. Premature apoptosis induction may disrupt viral release and dissemination of virus particles, thereby compromising LSDV replication. These findings suggest that \u003cem\u003eOrf132\u003c/em\u003e may modulate cell stress to optimize viral fitness.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLumpy skin disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSDV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLumpy skin disease virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGTPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egoatpox virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eopen reading frames\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCaPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCapripoxvirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esheeppox virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASFV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAfrican swine fever virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWest Nile virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHEV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatitis E virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eunfolded protein response\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHpi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehours post infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eeIF2α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eeukaryotic initiation factor 2α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch4\u003eCompeting interests\u003c/h4\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003ch4\u003eFunding\u003c/h4\u003e\n\u003cp\u003eThis work was supported by the Liaoning Provincial Department of Education project(JYTYB2024068).\u003c/p\u003e\n\u003ch4\u003eEthics approval and consent to participate\u003c/h4\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch4\u003eAuthors' contributions\u003c/h4\u003e\n\u003cp\u003e\u003cstrong\u003eYongtao Wang\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e, conceptualization, methodology, formal analysis, investigation, and writing-original draft;\u0026nbsp;\u003cstrong\u003eShiwei Zhang\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e, formal analysis, data curation, and visualization;\u0026nbsp;\u003cstrong\u003eHailing Li.\u003c/strong\u003e, methodology and resources;\u0026nbsp;\u003cstrong\u003eYali Feng\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e, investigation and resources;\u0026nbsp;\u003cstrong\u003eYing Zhang\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e, project administration, conceptualization, supervision, and writing-review and editing.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eAuthors' information\u003c/h4\u003e\n\u003cp\u003eKey Laboratory of Livestock Infectious Diseases, Ministry of Education, and Key Laboratory of Ruminant Infectious Disease Prevention and Control (East), Ministry of Agriculture and Rural Affairs, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, 120 Dongling Road, Shenyang 110866, China\u003c/p\u003e\n\u003cp\u003eYongtao Wang, Shiwei Zhang, Hailing Li, Yali Feng and Ying Zhang\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eManual OTJBBO. 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Mol Cell Biol. 1998;18(12):7499\u0026ndash;509.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Xu L, Gillette TG, Jiang X, Wang ZV. The unfolded protein response in ischemic heart disease. J Mol Cell Cardiol. 2018;117:19\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA. 2004;101(31):11269\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11(4):381\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, Nishimura K, Matsuo S. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology open. 2013;2(10):1084\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCirone M. ER Stress, UPR Activation and the Inflammatory Response to Viral Infection. Viruses 2021, 13(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGavil\u0026aacute;n E, Medina-Guzman R, Bahatyrevich-Kharitonik B, Ruano D. Protein Quality Control Systems and ER Stress as Key Players in SARS-CoV-2-Induced Neurodegeneration. Cells 2024, 13(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan J, Liu Y, Li W, Zhang Y, Chen G, Fang Y, He X, Jing Z. Lumpy Skin Disease Virus Infection Activates Autophagy and Endoplasmic Reticulum Stress-Related Cell Apoptosis in Primary Bovine Embryonic Fibroblast Cells. Microorganisms 2023, 11(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G. Viral control of mitochondrial apoptosis. PLoS Pathog. 2008;4(5):e1000018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedigeshi GR, Lancaster AM, Hirsch AJ, Briese T, Lipkin WI, Defilippis V, Fr\u0026uuml;h K, Mason PW, Nikolich-Zugich J, Nelson JA. West Nile virus infection activates the unfolded protein response, leading to CHOP induction and apoptosis. J Virol. 2007;81(20):10849\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohn L, Thomas S, Herchenr\u0026ouml;der O, P\u0026uuml;tzer BM, Schaefer S. Hepatitis E virus ORF2 protein activates the pro-apoptotic gene CHOP and anti-apoptotic heat shock proteins. PLoS ONE. 2011;6(9):e25378.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lumpy skin disease virus, Orf132, gene deletion, ER stress, reduced replication","lastPublishedDoi":"10.21203/rs.3.rs-6261646/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6261646/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLumpy skin disease (LSD), caused by lumpy skin disease virus (LSDV), is an emerging infectious disease in China that primarily affects cattle. LSDV and goatpox virus (GTPV) belong to the Capripoxvirus genus and exhibit high genomic homology, enabling cross-immunogenicity. Comparative genome analysis revealed that LSDV contains a unique gene, \u003cem\u003eOrf132\u003c/em\u003e, whose function remains uncharacterized. In this study, we first confirmed that the recombinant ORF132 protein exhibits immunoreactivity against sera from LSDV-infected cattle and GTPV-vaccinated cattle, and this cross-reactivity excluded the possibility of using the ORF132 protein to distinguish between LSDV and GTPV. To investigate the biological role of \u003cem\u003eOrf132\u003c/em\u003e, we generated an \u003cem\u003eOrf132\u003c/em\u003e deletion strain (LSDV-Δ\u003cem\u003eOrf132\u003c/em\u003e). Compared with that of the wild-type LSDV, the replication capacity of LSDV-Δ\u003cem\u003eOrf132\u003c/em\u003e was reduced approximately tenfold, indicating that \u003cem\u003eOrf132\u003c/em\u003e is critical for viral replication. Transcriptomic analysis of infected MDBK cells revealed significant alterations in Endoplasmic reticulum(ER) protein processing and unfolded protein response(UPR) pathways following \u003cem\u003eOrf132\u003c/em\u003e deletion. RT-qPCR validation showed marked upregulation of ER stress markers including \u003cem\u003eGrp78\u003c/em\u003e, \u003cem\u003eChop\u003c/em\u003e, and \u003cem\u003eGadd34\u003c/em\u003e. Subsequent apoptosis assays established that \u003cem\u003eOrf132\u003c/em\u003e deletion triggers CHOP-Caspase12-mediated apoptotic pathways. This dysregulated stress response cascade culminates in premature apoptotic scenarios, possibly resulting in a weakening of viral replication. Our findings collectively revealed that \u003cem\u003eOrf132\u003c/em\u003e is a critical gene for LSDV replication, plays an essential role in the virus's life cycle, and its deletion significantly impairs viral replication while inducing ER stress-related apoptosis.\u003c/p\u003e","manuscriptTitle":"Orf132: A Critical Gene for LSDV Replication and Its Role in ER Stress-Related Apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 06:04:42","doi":"10.21203/rs.3.rs-6261646/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-13T14:28:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T20:11:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T13:57:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T10:00:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148174332744058331886437759844776276659","date":"2025-03-31T12:20:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187095873756451993184131124240210271349","date":"2025-03-31T00:52:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18914170066944860044330349906502984774","date":"2025-03-29T12:53:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T19:47:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T05:35:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T05:12:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2025-03-19T12:30:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c41168ac-3500-49a0-9aa9-bff7cd09c47d","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:00:19+00:00","versionOfRecord":{"articleIdentity":"rs-6261646","link":"https://doi.org/10.1186/s12985-025-02813-8","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2025-05-30 15:57:17","publishedOnDateReadable":"May 30th, 2025"},"versionCreatedAt":"2025-03-24 06:04:42","video":"","vorDoi":"10.1186/s12985-025-02813-8","vorDoiUrl":"https://doi.org/10.1186/s12985-025-02813-8","workflowStages":[]},"version":"v1","identity":"rs-6261646","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6261646","identity":"rs-6261646","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}