Construction and Biological Characterization of ORF133-Deletion Mutant of Lumpy Skin Disease Virus

TL;DR

Researchers deleted a unique gene in the Lumpy Skin Disease Virus to study its role in viral replication and host adaptation.

Contribution

The study introduces a recombinant virus with a deleted ORF133 gene and mouse antibodies to investigate its role in LSDV.

Findings

ORF133 deletion affects viral replication in LSDV.

Mouse polyclonal antibodies against ORF133 were successfully generated.

The ORF133-deleted recombinant virus was constructed using homologous recombination.

Abstract

Lumpy skin disease virus (LSDV), a Capripoxvirus genus member, causes severe cattle disease. Though Capripoxviruses share high nucleotide sequence homology indicating common ancestry, they have evolved distinct host adaptations. The LSDV genome encodes numerous proteins, with ORF133 being LSDV-specific and lacking clear homologs in other Capripoxviruses, implying potential roles in host range and virulence. To explore ORF133’s function, this study generated mouse polyclonal antibodies against ORF133 and constructed the ORF133-deleted recombinant virus (LSDVΔORF133-EGFP) via homologous recombination with an EGFP reporter. Preliminary characterization showed that ORF133 deletion affects viral replication. This study provides critical tools and theoretical references for subsequent investigations into the functional mechanisms underlying ORF133 in LSDV.

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Full text

1. Introduction

Lumpy skin disease (LSD) is an acute, subacute, or chronic infectious disease in cattle caused by the infection of lumpy skin disease virus (LSDV). This disease was also formerly known by several synonyms, including Neethling poxvirus disease, bovine nodular eruption, and pseudo-urticaria. LSDV affects cattle of all ages and breeds, with dairy cows in peak lactation and calves being the most susceptible to infection [1]. Cattle herds infected with LSD exhibit impaired reproductive function and reduced productivity, which often manifested as abortion in pregnant cows, decreased milk production, infertility in bulls, and emaciation in infected cattle. Additionally, secondary bacterial infections are common, which can lead to cattle death in severe cases [2,3]. Furthermore, the disease can cause fever in cattle, as well as the widespread appearance of nodules or ulcers on the skin or internal organs [4,5]. The global outbreaks of LSD pose a significant threat to the healthy and stable development of the cattle industry and affect the international trade of animals and animal products [6]. Therefore, the World Organization for Animal Health (WOAH) classifies LSD as a notifiable animal disease required by law.

LSDV is a poxvirus with double-stranded enveloped DNA approximately 151 kb in size. It has only one serotype and no hemagglutinating activity and belongs to the family Poxviridae and the genus Capripoxvirus (CaPV) [7]. Its genomic sequence shares more than 96% homology with that of Goat Pox Virus (GTPV) and Sheep Pox Virus (SPPV) [5,8]. The LSDV genome encodes a large number of proteins, including 156 open reading frames (ORFs) [9]. These encoded proteins are involved in viral DNA replication, transcription, translation, nucleotide metabolism, structural assembly and stability, host range, and virulence [9]. By referring to the gene functions of other previously studied poxviruses, the functions of some LSDV-encoded proteins have been predicted. For example, proteins such as LSDV ORF039, LSDV ORF077, LSDV ORF083, and LSDV ORF139 are involved in viral DNA replication or may be essential for viral replication; proteins including LSDV ORF005, LSDV ORF006, LSDV ORF007, LSDV ORF136, and LSDV ORF142 may be associated with the regulation or immune evasion of LSDV [9]. Of note, the functions of some of these predicted proteins have been experimentally validated [10,11,12]. However, most of the current understanding of the aforementioned LSDV-encoded proteins is derived from the reference to or inference from known research findings on other members of the Poxviridae family. This underscores the necessity of conducting in-depth research on LSDV at present.

The full length of the LSDV ORF133 gene is 534 bp, encoding a protein of 177 amino acids (Supplementary File S1, Figure S1A,B). Analysis using online bioinformatics tools indicated that ORF133 is a hydrophilic protein with a stable structure, transmembrane domains, and a signal peptide. Its theoretical isoelectric point is 5.12. Among the potential phosphorylation sites, eight serine (Ser) residues, three threonine (Thr) residues, and two tyrosine (Tyr) residues have scores exceeding the threshold, suggesting that they may serve as phosphorylation sites for protein kinases [13,14] (Supplementary File S1, Figure S1C–F). In addition, homology analysis of gene sequences via NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, Nucleotide BLAST (Accessed on: 9 January 2025)) showed that the highest sequence homology of LSDV ORF133 with other poxviruses is only 83.75%—a significant discrepancy compared to the over 96% overall genomic sequence homology between LSDV and Goat Pox Virus (GTPV) or Sheep Pox Virus (SPPV). Furthermore, no homologous results were obtained in the alignment of homologous amino acid sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi, Protein BLAST (Accessed on: 10 January 2025)), indicating that the protein encoded by the ORF133 gene is involved in the unique biological functions of LSDV (Supplementary File S2). In this study, the ORF133 gene (gene location: 119773-120306) from the LSDV/FJ/CHA/2021 strain (GenBank accession number: OP752701.1) was used as the research target. A mouse-derived polyclonal antibody against ORF133 with strong reactivity and high titer was prepared. Meanwhile, a transfer vector containing the enhanced green fluorescent protein (EGFP) gene was constructed. Homologous recombination followed by multiple rounds of purification yielded a homozygous recombinant virus, whose biological characteristics were analyzed. This work provides a critical material basis and technical support for investigating the biological function of LSDV ORF133, as well as for clarifying the role of this unique gene in understanding the interaction between LSDV and its host, and the host specificity of poxviruses.

2. Materials and Methods

2.1. Viral Strains, Cell Lines, Vectors, Bacterial Strains, and Animals

The LSDV/FJ/CHA/2021 strain (GenBank: OP752701) was isolated and stored in the biosafety level-3 (BSL-3) laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (LVRI CAAS), and the LSDVΔORF133-EGFP strain was constructed and purified by our laboratory. LSDV/FJ/CHA/2021 is denoted by LSDV or WT-LSDV in the text; human embryonic kidney 293T (HEK-293T) cells, Lamb testicular (LT) cells, baby hamster kidney (BHK) cells, Verda reno (Vero) cells, Porcine Kidney-15 (PK) cells, F81 cells (feline kidney cell line), Mardin Darby Bovine Kidney (MDBK) cells, and bovine mammary epithelial cells (BMEC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BBI, E600003-0500, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; CELL-BOX, AUS-01S-02, Changsha, China) and 1% penicillin–streptomycin (Shanghai Epizyme Biomedical Technology Co., Ltd., CB010, Shanghai, China) at 37 °C with 5% CO_2_. All the aforementioned cells are preserved in our laboratory. The homologous recombination vector pUC-19T was preserved in our laboratory at the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences [15]. The prokaryotic expression vector pET-28a(+) was purchased from Miaolingbio (MiaoLing Plasmid Platform, Cat. No. P0023, Wuhan, China). Competent cells from Escherichia coli (E. coli) BL21 (DE3) and E. coli DH5α were purchased from Bioengineering Co., Ltd. (Shanghai, China). All the aforementioned viruses and cells were tested for mycoplasma, with negative results. BALB/c mice were purchased from the Laboratory Animal Center of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

2.2. Main Reagents

The main reagents used in this experiment are presented in Table 1.

2.3. Bioinformatics Analysis of LSDV ORF133

Based on the gene/protein sequence of LSDV/FJ/CHA/2021 (GenBank: OP752701.1), the homology of LSDV-encoded gene/protein sequences was analyzed individually using the online analysis function of NCBI BLAST [18]. The highest homology value of each sequence was collected for final statistical analysis. The ORF133 protein sequences of different LSDV strains were retrieved from NCBI, and the conservation of ORF133 protein sequences was analyzed via the DNAMAN software (LynnonBiosoft, San Ramon, CA, USA) (Version 9.0) [19]. The physicochemical properties of the ORF133 protein were analyzed using the online ProtParam tool in ExPasy (https://web.expasy.org/protparam (Accessed on: 15 January 2025)). TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM2.0/ (Accessed on: 15 January 2025)) was employed to predict the transmembrane structure of ORF133, while SignalP 4.1 (https://www.cbs.dtu.dk/services/SignalP/ (Accessed on: 16 January 2025)) was used to predict its signal peptide. The hydrophilicity and hydrophobicity of the ORF133 protein were analyzed using the online ProtScale tool in ExPasy. The NetPhos 3.0 online software (http://www.cbs.dtu.dk/services/NetPhos (Accessed on: 20 January 2025)) was utilized to predict the phosphorylation sites of the ORF133 protein [20].

2.4. Construction of the Prokaryotic Expression Vector pET28a-ORF133

Based on the ORF133 gene sequence of the LSDV/FJ/CHA/2021 strain, a pair of specific primers (Table 2, Primer1) was designed using the SnapGene software (GSL Biotech LLC, Boston, MA, USA) (Version 8.1.1) [21]. Polymerase chain reaction (PCR) amplification was performed with the following components: DNA extracted from this strain as the template (10 pg–30 ng), 2 μL each of the upstream and downstream primers of Primer1 (5.45 nmol/OD), 25 μL of 2× Phanta Max Master Mix, and deionized water added to make up the total volume to 50 μL. The PCR cycling program was set as follows: initial denaturation at 95 °C for 1 min; followed by 34 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and extension at 72 °C for 1.5 min; a final extension step at 72 °C for 5 min; and subsequent cooling to 12 °C. The PCR products were analyzed by 1.5% agarose gel electrophoresis, and the target band was recovered for subsequent use. For double-enzyme digestion, the reaction system was prepared with 1 μg of the pET-28a+ vector and the above-mentioned gel-extracted product fragment as templates, 5 μL of 10× NEBuffer, 1 μL each of the HindIII and EcoRI Restriction Enzymes, and nuclease-free water added to a final volume of 50 μL. The prepared reaction mixture was gently mixed by pipetting up and down, briefly centrifuged, then incubated at 37 °C for 15 min, followed by heat inactivation at 65 °C for 20 min. The target band of the enzyme-digested products was recovered by gel extraction, and ligation was carried out overnight at 16 °C using T4 DNA ligase. The ligation product was transformed into E. coli BL21 (DE3) competent cells, which were then spread on solid LB medium plates (100 μg/mL kanamycin) and cultured overnight at 37 °C. Single colonies were then picked and cultured with shaking, followed by plasmid extraction from the bacterial solution. After PCR identification, the positive plasmid was named pET28a-ORF133. All primer sequences in this study are presented in Table 2.

2.5. Induction, Expression, and Purification of the Target Protein

The original bacterial solution of plasmid pET28a-ORF133 was cultured in the LB medium (100 μg/mL kanamycin) at a ratio of 1:100. When the OD600 value reached 0.81.0, its expression was induced with 1 mmol/L IPTG, and the bacterial solution was incubated in a shaker at 37 °C for another 68 h. After centrifugation, the bacterial pellet was retained, resuspended in 1× PBS, and then disrupted by ultrasonication. Following re-centrifugation, the supernatant and precipitate were collected separately, mixed with 4× protein loading buffer, and heated in a metal bath at 100 °C for 10 min. The expression form of the target protein was analyzed by SDS-PAGE electrophoresis, with Coomassie Brilliant Blue used for staining. After confirming the optimal expression conditions, the target protein was induced in large quantities and purified using a protein purification kit (Tiangsa, 220952-5, Beijing, China). The purified target protein was mixed with 4× protein loading buffer, heated in a metal bath at 100 °C for 10 min, and subjected to SDS-PAGE analysis to verify the purification efficiency of the target protein.

2.6. Preparation and Titer Determination of Polyclonal Antibodies

For the primary immunization, the purified ORF133 protein was mixed with the GEL adjuvant (accounting for 15–18% of the total volume), and 6-week-old female BALB/c mice were subcutaneously immunized at a dose of 200 μg per mouse via the dorsal route. For the secondary immunization, the target protein was thoroughly mixed with the GEL adjuvant (accounting for 20–25% of the total volume) and administered at the same dose of 200 μg per mouse. For the third and fourth immunizations, the purified target protein alone was used for immunization at a dose of 200 μg per mouse, with an interval of 7 days between consecutive immunizations. On the 7th day after the completion of the four-round immunization protocol, blood samples were collected from the orbital sinus, followed by serum separation and collection. Meanwhile, the negative serum samples collected prior to the primary immunization were used as the control group.

The antibody titer in serum was determined by the indirect enzyme-linked immunosorbent assay (ELISA). (1) Coating: ELISA microplates were coated with the purified target protein ORF133 at 0.005 μg/mL (100 μL/well, 3 replicate wells) and incubated overnight at 4 °C; (2) Blocking: The plates were blocked with a blocking buffer (50 g/L skimmed milk powder, 100 μL/well, 3 replicate wells) at 37 °C for 1 h; (3) Primary antibody: The ORF133 polyclonal antibody was serially diluted at ratios from 1: 400 to 1: 102,400 (100 μL/well, 3 replicate wells) and incubated at 37 °C for 1 h, with the pre-immunization serum used as the negative-control serum; (4) Secondary antibody: The Goat Anti-Mouse IgG-FITC antibody (1:4000 dilution for ORF133) was incubated at 37 °C for 1 h; (5) Substrate reaction: The TMB substrate solution (100 μL/well) was added, and the plates were incubated at 37 °C for 8 min in the dark. Finally, a stop solution (50 μL/well) was added, and the OD450 values were immediately measured using a multifunctional microplate reader. Sera from non-immunized animals were used as negative controls. The antibody titer of the test serum was defined as the dilution at which the ratio of the OD450 value of the test serum to that of the negative control (P/N ratio) was greater than 2.1.

2.7. SDS-PAGE and Western Blotting

Purified target protein samples, HEK-293T cell samples transfected with the target plasmid, or MDBK cell samples infected with LSDV at different time points were mixed thoroughly with 4× protein loading buffer, heated in a metal bath at 100 °C for 10 min, and then subjected to SDS-PAGE electrophoresis. The proteins were transferred onto a methanol-activated PVDF membrane. After blocking with a blocking buffer (TBST containing 5% skimmed milk powder) at room temperature for 1 h, the excess PVDF membrane was cut off according to the position of the protein marker. The corresponding primary antibody was added, and incubation was carried out at 4 °C for 8–10 h. After incubation, the PVDF membrane was washed with TBST buffer, followed by the addition of the corresponding secondary antibody and incubation at room temperature for 1 h. Finally, a chemiluminescent reagent (ECL) was used to visualize and analyze the results in a gel imaging system (Model: GE-AI600, Boston, MA, USA).

2.8. Indirect Immunofluorescence Assay (IFA)

MDBK cells infected with the LSDV/FJ/CHA/2021 strain or HEK-293T cells transfected with the target plasmid (Pcaggs-ORF133-Flag) were cultured in specialized laser confocal dishes (Nest, 801001, Wuxi, China). At the predetermined time point, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.5% Triton X-100 at room temperature for 5 min, and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. The corresponding labeled antibody (anti-Flag) or the prepared mouse-derived ORF133 polyclonal antibody was added as the primary antibody, followed by incubation at 4 °C overnight. Subsequently, in a dark environment, the corresponding mouse-derived or rabbit-derived fluorescent dye-conjugated secondary antibody (Table 1) was added and incubated at 37 °C for 30 min. In a dark environment, the corresponding fluorescent dye-conjugated secondary antibody was added and incubated at 37 °C for 30 min. Finally, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The cell culture dish was placed under a laser confocal microscope (Model: ZEISS, Oberkochen, Germany) to observe the subcellular localization of the target protein.

2.9. Construction of the pUC-19T-LSDVΔORF133-EGFP Transfer Vector

On the basis of the modified early–late H5 promoters (pmH5) of the vaccinia virus (VACV) established in our laboratory in previous studies, the Cre-loxP cassette system was successfully constructed in this research for the sequential deletion of target genes in the viral genome [15]. According to the ORF133 gene sequence of the LSDV/FJ/CHA/2021 strain, the left-arm primer pair (Primer2) and right-arm primer pair (Primer3) for ORF133 homologous recombination were designed using the SnapGene software. Taking the extracted DNA of the LSDV/FJ/CHA/2021 strain as the template, high-fidelity DNA polymerase and the aforementioned primers were used for PCR amplification of the left and right homologous arms. Using plasmid pUC-19T as the template, Primer4 was employed to amplify the EGFP gene for the transfer vector [15]. The PCR system was prepared following the manufacturer’s instructions. After verifying the PCR amplicons by 1.0% agarose gel electrophoresis, the target fragments were recovered using a gel extraction kit, and their nucleic acid concentrations were determined. Meanwhile, the correctly identified left and right homologous arms and the EGFP target fragment were subjected to restriction enzyme digestion, followed by fusion ligation using T4 DNA ligase. After validating the ligation product via 1.0% agarose gel electrophoresis, the target fragment was recovered by gel extraction, and its nucleic acid concentration was measured. The pUC-19T vector was double-digested with EcoRI and HindIII; then the cloned fragments (left homologous arm, EGFP expression cassette, and right homologous arm) were ligated into the digested pUC-19T vector using T4 DNA ligase. The ligation product was mixed thoroughly with E. coli DH5α competent cells, followed by transformation procedures that included heat shock treatment (water bath at 42 °C for 60 s) and incubation in a sterile antibiotic-free LB medium. The resultant mixture was then spread onto solid ampicillin-containing 2YT medium plates and cultured overnight at 37 °C. Single colonies were picked for liquid culture, and after PCR identification, plasmids were extracted using a plasmid miniprep kit and sent to Tsingke Biotechnology Co., Ltd. (Beijing, China) for sequencing. The positive plasmid was named pUC-19T-LSDVΔORF133-EGFP.

2.10. Construction and Purification of the ORF133-Deleted Recombinant LSDV

When Vero cells in 6-well plates were in good growth status with a confluency of 70–80%, the pUC-19T-LSDVΔORF133-EGFP plasmid was transfected into Vero cells using the Lipo8000™ transfection reagent (C0533-7.5 ml, Shanghai, China). After 12 h, the cells were infected with 2 μL of LSDV/FJ/CHA/2021 (TCID_50_ = 10^−7.5^ mL^−1^). At 24 h post-infection (hpi), the cytopathic effect (CPE) and green fluorescent signals were observed under a fluorescence microscope. Approximately 72 h later, the cell–virus mixture was harvested, and cell debris was removed by centrifugation. The viral supernatant was collected, stored at −80 °C for future use, and named LSDVΔORF133-EGFP.

Three methods were mainly used for the purification of the LSDVΔORF133-EGFP recombinant virus: (1) Serial Dilution: When the confluency of Vero cells in 96-well plates exceeded 80%, the collected virus solution was serially diluted 10-fold with DMEM (containing 10% FBS, without antibiotics). Each well was infected with 100 μL of the serially diluted virus, and the cytopathic effect (CPE) and fluorescent signals were observed and recorded. After 4 days, the 96-well plate was sealed with parafilm and subjected to three repeated freeze–thaw cycles at −80 °C; then the virus solution from the fluorescent well with the highest dilution factor was collected in a sterile environment and stored at −80 °C for further purification and passage. (2) Single-Clone-Cell Picking: For the virus passaged by serial dilution, the fluorescent well with the highest dilution factor, appropriate fluorescence intensity, and good cell status was selected for single-clone-cell picking. The medium in the fluorescent well was aspirated and discarded using a pipette, and a trypsin digestion solution was added for digestion. The cell digestion status was observed under a microscope; once the cells detached, they were immediately resuspended and diluted with DMEM (containing 10% FBS, without antibiotics) and transferred to a 1.5 mL centrifuge tube pre-filled with 1.5 mL of DMEM (containing 10% FBS, without antibiotics). Subsequently, the entire solution was transferred to a 35 mm cell culture dish. After approximately 1 min to allow the cells to settle slightly, the culture dish was placed under a fluorescence microscope to locate single fluorescent cells. A 2.5 μL pipette was adjusted to 1 μL to aspirate individual fluorescent cells (ensuring as much as possible that only a single fluorescent cell was collected) and transferred to a 1.5 mL centrifuge tube containing 800 μL of DMEM (containing 10% FBS, without antibiotics). For one round of purification, 10–20 single fluorescent cells were collected, subjected to two repeated freeze–thaw cycles, and then stored at −80 °C for later use. If a large number of non-fluorescent cells were aspirated, the above process of picking single-clone cells from the culture dish could be repeated. (3) Plaque Assay: When the confluency of Vero cells in 12-well plates exceeded 90%, the collected virus from single fluorescent cells was used to infect the cells. After observing CPE and fluorescent signals for 3 days, wells with large, bright, and single fluorescent foci were selected for the plaque assay. The cell culture medium in the 12-well plate was discarded, and each well was added with 2 mL of a mixture of 2% low-melting-point agarose (preheated in a 60 °C water bath for 30 min before the experiment) and 2× DMEM at a ratio of 1:1. The plate was left at room temperature for 10 min to allow the mixture to solidify, then transferred to a cell incubator (Thermo Scientific, 51023126, Waltham, MA, USA) (37 °C, 5% CO_2_) for further culture. After viral plaques formed, the fluorescent positions were observed and marked under a fluorescence microscope. A single fluorescent plaque was picked using the tip of a 1 mL pipette, transferred to a 1.5 mL centrifuge tube containing 800 μL of DMEM (containing 10% FBS, without antibiotics), subjected to two repeated freeze–thaw cycles, and stored at −80 °C for later use. During the purification process, the above three methods were alternately used as needed. After 10 generations of purification, the virus culture was scaled up every two generations, and viral genomic DNA was extracted for PCR identification to confirm the success of virus purification.

2.11. Identification of the LSDVΔORF133-EGFP Recombinant Virus

PCR Identification: After expanding the culture of the purified recombinant virus on MDBK cells, viral genomic DNA was extracted. The genomic DNA of the parental strain (WT-LSDV) was used as a control, and PCR identification was performed with Primer2-133LF/Primer3-133RR and Primer5, as listed in Table 2. When identification was performed with the primer pair Primer2-133LF/Primer3-133RR, the simultaneous appearance of target bands at approximately 2700 bp (for the recombinant strain) and 2100 bp (for the parental strain) compared with the parental strain (WT-LSDV) indicated that the recombinant virus had not been purified successfully. In contrast, the amplification of only the ~2700 bp target band verified the successful purification of the recombinant virus. For identification with Primer Pair 5, the presence of a ~470 bp target band in the recombinant virus relative to the parental strain (WT-LSDV) denoted unsuccessful purification, whereas the absence of this band confirmed successful purification of the recombinant virus.

Sequencing Identification: The extracted genomic DNA of the recombinant virus and the parental strain (WT-LSDV) was sent to Tsingke Biotechnology Co., Ltd. (Beijing, China) for sequencing. The upstream and downstream primers for sequencing were Primer2-133LF/Primer3-133RR, as shown in in Table 2, and the sequencing results were compared with the designed theoretical sequence.

Western Blotting Verification: MDBK cells were infected with the purified recombinant virus, with MDBK cells infected with the parental strain WT-LSDV and uninfected MDBK cells as controls. The polyclonal antibody against ORF133 (1:5000), the monoclonal antibody against ORF29 (1:5000), and the antibody against GFP (1:5000) were used as primary antibodies. GAPDH (1:8000) was used as the internal reference, and HRP-labeled goat anti-BALB/c mouse IgG (1:10,000) was used as the secondary antibody. After visualization by ECL chemiluminescence, Western Blotting analysis was performed.

2.12. Titration of the LSDVΔORF133-EGFP Recombinant Virus

When MDBK cells in 96-well cell culture plates reached 80–90% confluence, the recombinant virus LSDVΔORF133-EGFP was serially diluted to gradients of 10-1 to 10-10. A total of 100 μL of the diluted virus solution was added to each well, with 8 replicate wells for each gradient. The 96-well culture plates were incubated in a 37 °C incubator with 5% CO_2_. The number of wells showing a cytopathic effect (CPE) was observed and recorded under a microscope. Continuous observation was conducted for 5 days to calculate the 50% tissue culture infective dose (TCID_50_). The titration process was repeated three times to ensure the accuracy of the titer results of the LSDVΔORF133-EGFP recombinant virus.

2.13. Viral Growth Kinetics

To compare the replication differences of WT-LSDV and LSDVΔORF133-EGFP, the growth kinetics of the two viruses were examined. A six-well plate was inoculated with about 1  × 10^5^ MDBK cells or BMECs. After incubation overnight, the cells were infected with WT-LSDV (10^7.515^ TCID_50_) or LSDVΔORF133-EGFP (10^5.85^ TCID_50_) at an MOI of 1. After incubation for 12, 24, 48, 72 and 96 h, the supernatant was collected, and the viral titer was measured as the median tissue culture infective dose (TCID_50_).

2.14. Genetic Stability of the Recombinant Virus

The purified recombinant virus LSDVΔORF133-EGFP was continuously cultured on MDBK cells from passage 20 (F20) to passage 35 (F35). Every 5 passages, the infection status and fluorescence of cells infected by the recombinant virus were observed and recorded. Meanwhile, viral genomic DNA after infection was extracted and subjected to PCR identification using Primer2-133LF/Primer3-133RR and Primer5, as listed in Table 2, with the genomic DNA of the parental strain as a control, to determine the genetic stability of the recombinant virus.

2.15. Morphological Characteristics of the Recombinant Virus

MDBK cells were passaged into T75 cell flasks. When the cell density reached approximately 80%, the cells were inoculated with the LSDV/FJ/CHA/2021 strain and the LSDVΔORF133-EGFP recombinant virus strain, respectively. On the 3rd day post-virus infection, the cytopathic effect (CPE) and fluorescence of the recombinant virus were observed to confirm good viral infection status. Subsequently, 10 mL of PBS was added to wash the cells twice, followed by cell digestion with trypsin. After terminating the digestion process, the cells were transferred to 1.5 mL centrifuge tubes and centrifuged at 1500 r/min for 10 min. The supernatant was discarded, and the remaining cells were those infected with the virus. A pre-cooled (4 °C) glutaraldehyde fixation mixture was gently added along the side wall of the EP tube for fixation, and the tubes were incubated overnight at 4 °C. On the next day, the samples were sent to the electron microscopy processing room for sample preparation and treatment. After the test samples were prepared, observation and photography were performed using a transmission electron microscope (HT770, Hitachi Electronics, Tokyo, Japan) in the central instrument room.

2.16. Screening of Suitable Cell Lines for the Growth of the Recombinant Virus

MDBK, BMEC, OTLC, Vero, BHK, PK, HEK-293T, and F81 cells were seeded into 35 mm dishes. When the cell density reached approximately 80%, the cells were infected with the recombinant virus LSDVΔORF133-EGFP or LSDV at the same viral dose. After 48 h, the fluorescence of the recombinant virus in each cell line was observed to determine the suitable cell lines for the growth of the LSDVΔORF133-EGFP recombinant virus.

2.17. Statistical Analysis

Statistical analysis was performed using the GraphPad Prism software (Insightful Science, San Diego, CA, USA) (Version 8.01). All values represent the mean of at least three independent experiments. The standard deviation (SD) and standard error of the mean (SEM) were calculated and determined from at least three technical replicates unless otherwise stated. One-way analysis of variance (ANOVA) was employed to analyze three or more group means [22]. Statistical significance was evaluated by determining the p-value using the two-tailed Student’s t-test (two-tailed distribution). (ns: non-significant, * p < 0.05 and ** p < 0.01).

2.18. Ethics Statemcnt

All animal experiments were performed in strict accordance with the relevant regulations specified in the Guide for the Care and Use of Laboratory Animals issued by the Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences (CAAS, Beijing, China). All experimental protocols involved in this study were reviewed and approved by the Animal Ethics Committee of LVRI, CAAS (Approval Document No.: SCXK (GAN) 2020-0010), with the assigned experimental ethics approval number of LVRIAEC-2025-090. Additionally, the standard experimental protocols adopted in this study were also endorsed by the Institutional Animal Care and Use Committee (IACUC) of LVRI, CAAS.

3. Results

3.1. LSDV ORF133 Is a Gene with Unknown Function

Homology analysis was performed on the 156 ORF gene sequences or amino acid sequences of the LSDV/FJ/CHA/2021 strain (GenBank accession number: OP752701.1) one by one using the NCBI database. It was found that the ORF133 gene sequence had the lowest homology of 83.75%, which is a gene with unknown function (Figure 1A). Moreover, the homology of the ORF133 amino acid sequence alignment was extremely low, with no known counterparts in the Poxviridae family (Figure 1B, Supplementary File S2). Conservation analysis of the ORF133 protein (WGU25513.1) using the DNAMAN software (LynnonBiosoft, San Ramon, CA, USA) (Version 9.0) showed that the ORF133 protein is relatively conserved among different LSDV isolates (Figure 1C, Supplementary File S3). These results indicate that ORF133 is a gene with unknown function, having no known counterparts in the Poxviridae family but being relatively conserved among different LSDV isolates.

3.2. LSDV ORF133 Exhibits Immunogenicity

To investigate the biological function of LSDV ORF133 and facilitate the subsequent identification of recombinant viruses, the LSDV ORF133 protein was expressed using a prokaryotic expression system and purified. The results are as follows: The target fragment of the LSDV ORF133 gene, with a size of approximately 530 bp, was amplified via PCR technology (Figure 2(Aa)). Double-enzyme digestion identification of the recombinant plasmid pET28a-ORF133 revealed a pET-28a (+) vector fragment of around 5000 bp (Figure 2(Ab)). The ORF133 protein was successfully expressed under induction and was mainly present in the precipitate (Figure 2B), indicating that the protein was predominantly expressed in the form of inclusion bodies. SDS-PAGE electrophoresis analysis of the purified ORF133 protein showed that its molecular weight was consistent with the expected band size, approximately 20 kDa (Figure 2C). Western Blotting results confirmed that the prepared LSDV ORF133 polyclonal antibody could specifically recognize the ORF133 protein (Figure 2D). Antibody titer determination demonstrated that the ORF133 protein could induce BALB/c mice to produce specific antibodies with high immunogenicity and titer, reaching up to 1:128,000 (Figure 2E). Indirect immunofluorescence assay (IFA) results showed that red fluorescent signals were observed in Madin-Darby bovine kidney (MDBK) cells infected with the LSDV/FJ/CHA/2021 strain, whereas no fluorescent signal was detected in the negative-control group, suggesting that the prepared mouse-derived anti-ORF133 polyclonal antibody could specifically recognize the ORF133 protein in LSDV (Figure 2F). Subcellular localization analysis indicated that bright punctate fluorescent signals were detected in the cytoplasm of both 293T cells transfected with the eukaryotic expression plasmid pcaggs-ORF133-Flag and MDBK cells inoculated with LSDV, confirming that the ORF133 protein is localized in the cytoplasm (Figure 2G). Western Blotting analysis of MDBK cells at different time points post-LSDV infection showed that the ORF133 protein band was approximately 25 kDa in size, and the protein could be detected as early as 12 h post-infection (hpi) (Figure 2H). Combining the above results, the successfully prepared mouse-derived polyclonal antibody against the ORF133 protein exhibits good specificity and favorable immunogenicity.

3.3. Construction, Purification, and Identification of ORF133 Deletion in the LSDV Strain

To investigate the function of ORF133 during the infection of lumpy skin disease virus (LSDV), a gene deletion transfer vector, pUC19T-LSDVΔORF133-EGFP, was constructed in this study. Subsequently, homologous recombination was induced between LSDV and the transfer vector plasmid DNA, thereby generating a novel viral strain deficient in the ORF133 gene (LSDVΔORF133-EGFP). The detailed construction procedure is illustrated in Figure 3A. Based on the schematic diagram, ORF133 was deleted from the wild-type LSDV/FJ/CHA/2021 strain (GenBank: 37336054) via homologous recombination (Figure 3A). Nucleic acid electrophoresis results showed that the fragment sizes of the amplified products of the left homology arm gene, the right homology arm gene, and the EGFP gene expression cassette were consistent with the bands expected (804 bp, 735 bp, and 1130 bp, respectively; Figure 3B). Subsequently, the above target fragments were fused and ligated, and the size of the amplified product after fusion of the three fragments was consistent with the expected size of approximately 2660 bp (Figure 3C). The above three fused fragments and the pUC-19T vector were double-digested with EcoRI and HindIII (Figure 3D(1–4)), and the recovered target fragments were then ligated with T4 DNA ligase to obtain the recombinant transfer vector plasmid pUC-19T-LSDVΔORF133-EGFP (Figure 3D(5–6)). As homologous recombination occurred between LSDV and the transfer vector plasmid DNA, a cytopathic effect (CPE) and green fluorescence were observed under a fluorescence microscope at 24 h post-infection. With the extension of LSDV infection time, the fluorescent foci gradually increased in brightness and number, and this recombinant virus was named LSDVΔORF133-EGFP (Figure 3E). The collected LSDVΔORF133-EGFP virus underwent 19 passages of purification and culture (Supplementary File S1, Figure S2A). Ultimately, PCR identification (Figure 3F), sequencing verification (Supplementary File S1, Figure S2B), and Western Blotting validation (Figure 3G) confirmed the successful purification of the recombinant virus, LSDVΔORF133-EGFP, obtained from the purification process. In short, following the construction of the transfer vector pUC19T-LSDVΔORF133-EGFP, the recombinant virus LSDVΔORF133-EGFP was successfully constructed using homologous recombination technology. The recombinant virus was purified by serial dilution, picking of monoclonal cells, and the plaque assay. Finally, PCR identification, Western Blotting analysis, and sequencing confirmed the successful acquisition of the purified LSDVΔORF133-EGFP recombinant virus.

3.4. Analysis of the Biological Characteristics of the LSDVΔORF133-EGFP Recombinant Virus

Growth Curve: Based on the recorded cytopathic effect (CPE) data of MDBK cells and BMECs, the recombinant strain and the wild-type strain exhibited comparable infection levels in both cell types. The TCID_50_ of the recombinant strain, LSDVΔORF133-EGFP, was calculated to be 10^4.5^ mL^−1^, whereas that of the wild-type strain was determined as 10^7.5^ mL^−1^. During virus passage, the replication plateau phase of the recombinant strain at harvest was observed at approximately 72 h post-inoculation. Meanwhile, the growth curve results demonstrated that the proliferative capacity of this recombinant strain was lower than that of its parental strain (Figure 4A). Screening of Susceptible Cell Lines: To investigate the susceptibility of LSDV to different host cells, we infected eight distinct cell lines with LSDVΔORF133-EGFP or LSDV-WT, respectively, and detected the proliferation of both viruses in cells from different species. All eight cell lines could be infected by the LSDVΔORF133-EGFP recombinant virus but with varying susceptibility. Bovine kidney epithelial (MDBK) cells, bovine mammary epithelial cells (BMECs), African green monkey kidney epithelial (Vero) cells, and lamb testicular (LT) cells all exhibited high susceptibility, suggesting that both LSDVΔORF133-EGFP and LSDV-WT may also transmit among species such as monkeys and sheep. In contrast, hamster-derived BHK cells, porcine-derived PK cells, human-derived 293T cells, and feline-derived F81 cells showed significantly lower susceptibility to LSDVΔORF133-EGFP or LSDV-WT compared with the aforementioned cells. However, LSDVΔORF133-EGFP displayed slightly higher susceptibility to porcine-derived PK cells, human-derived 293T cells, and feline-derived F81 cells than LSDV-WT, highlighting that the ORF133 gene is highly likely associated with host selectivity during LSDV infection, though this hypothesis requires further verification. Furthermore, it should be emphasized that both LSDVΔORF133-EGFP and LSDV-WT exhibited extremely high infectivity towards MDBK cells and BMECs compared with cells derived from monkeys, sheep, hamsters, pigs, and cats, which also explains the key reason why LSDV can infect cattle and cause disease (Figure 4B). Genetic Stability: The purified recombinant virus LSDVΔORF133-EGFP was serially passaged to the 35th generation (F35) on MDBK cells. Fluorescence observation showed that the recombinant virus could stably inherit and correctly express the green fluorescent protein, and PCR results confirmed no mutations or reversion events in the recombinant virus, demonstrating good genetic stability of LSDVΔORF133-EGFP (Figure 4C). Morphological Characteristics: Transmission electron microscopy (TEM) observations revealed that mature LSDV virions possess a biconcave core, two lateral bodies, and an outer cellular envelope. The latter is barrel-shaped, containing virus-specific proteins within the envelope, and the outer surface of the envelope is wrinkled, forming irregular protrusions. The biconcave core inside the virion is dumbbell-shaped, where the viral genome is concentrated. The outer wall of the core consists of two protein outer membranes: the inner membrane is continuous with a certain number of channels, and its thickness and density are similar to those of the plasma membrane; the outer membrane has a fence-like structure with nail-shaped projections embedded in the inner membrane. No significant morphological changes were observed in the virion size (approximately 230 nm × 280 nm), outer capsid, or central chromatin between LSDVΔORF133-EGFP and LSDV-WT virions (Figure 4D). Based on above results, ORF133 may have an important impact on the proliferation and replication of LSDV.

4. Discussion

Lumpy skin disease virus (LSDV), Sheep Pox Virus (SPPV), and Goat Pox Virus (GTPV) all belong to the genus Capripoxvirus and can cause diseases in cattle, sheep, and goats, respectively [23,24]. Interestingly, however, although the three viruses share over 96% overall nucleotide identity among their genomes and exhibit highly similar clinical symptoms, they display distinctly different host ranges. Clearly, this host range specificity is closely associated with the differential genes between the viruses [25,26,27]. Furthermore, it should be emphasized that LSDV is a double-stranded DNA virus with a highly stable genome, a low probability of genetic variation, and only one serotype [28,29]. However, recent studies have demonstrated that LSDV strains isolated from different regions exhibit significant diversity in single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), while novel recombinant-like vaccine strains have also been identified in Asia [30,31,32]. In this study, the LSDV/FJ/CHA/2021 strain (GenBank: OP752701) was used as the research subject, and homology analysis of the sequences of its 156 ORFs (open reading frames)—including both gene (nucleotide) sequences and amino acid sequences—was performed using the NCBI database. The results revealed that ORF133 is the “Specific gene” with the lowest homology; crucially, the ORF133 gene is highly conserved across different LSDV isolates (Figure 1A–C; Supplementary Files S2 and S3). Based on this observation, fully deciphering the function of the ORF133 gene plays a crucial role in helping us understand its involvement in LSDV virulence, host range, and genetic evolution.

To date, the function of the ORF133 gene (ORF132 in other LSDV strains) of lumpy skin disease virus (LSDV) has only been superficially linked to LSDV replication and cellular apoptosis, and its function has not been fully characterized—primarily due to the lack of species-specific research tools, particularly antibodies targeting this protein [33]. In this study, obtaining a high yield of protein suitable for immunization was the primary prerequisite for polyclonal antibody preparation, and induction with IPTG significantly increased the production of the ORF133 protein (Figure 2B). In addition, purifying high-concentration proteins from crude extracts for immunization was also a critical step. A well-established protein purification kit was employed in this experiment, which not only simplified the purification procedure but also effectively improved the purity of the target protein (Figure 2C). Immunogenicity refers to the ability of a protein to induce the body to produce specific antibodies, with evaluation criteria mainly including antibody titer and antibody specificity. The indirect ELISA method was used to evaluate the protein in this study. In comparison, direct ELISA exhibits weak signals and is susceptible to background interference, making it difficult to accurately distinguish dilution gradients of low-concentration antibodies, whereas competitive ELISA is more suitable for detecting antigens rather than antibody titers [34] (Figure 2E). For the evaluation of antibody specificity, Western Blotting and immunofluorescence assay (IFA) confirmed that the antibodies only bound to the target protein and did not cross-react with impurity proteins or host cell proteins [35] (Figure 2D,F,G). Notably, these antibodies were successfully applied to the identification of ORF133 gene-deleted recombinant viruses, confirming that the LSDVΔORF133-EGFP recombinant virus had been successfully purified (Figure 3G).

Homologous recombination refers to the process of genetic material exchange that occurs between DNA fragments with high sequence similarity. The use of homologous recombination to construct DNA recombinant viruses is the most common method, where reporter genes are used to replace viral virulence genes, such as the enhanced green fluorescent protein (EGFP). A variety of recombinant viruses have been successfully constructed and established through homologous recombination, including African swine fever virus (ASFV) [36], herpes simplex virus type 1 (HSV-1) [37], and LSDV [38]. LSDV has a large genome encoding numerous genes. By constructing virus strains with a specific single-gene deletion and comparing their biological characteristics with those of the wild-type strain, the role of the target gene in the LSDV life cycle can be revealed [11,12,39,40]. In this study, homologous recombination was used to replace the ORF133 gene in the wild-type LSDV (WT-LSDV) strain with an EGFP reporter gene, generating the recombinant virus LSDV-∆ORF133-EGFP. It has been reported that LSDV can replicate in cell lines such as lamb testis (LT) cells, lamb kidney (LK) cells, bovine kidney (MDBK) cells, and ovine testis (OA3.TS) cells, causing cytopathic effects and the formation of intracellular inclusion bodies [41,42]. Additionally, inserting EGFP into the LSDV genome does not affect viral growth performance, and the fluorescence intensity of EGFP can be used to directly monitor the replication of the reporter virus [38]. In this study, the LSDV-∆ORF133-EGFP strain was inoculated into eight different well-maintained cell lines preserved in our laboratory. The results showed that the ORF133-deleted strain grew extremely well in MDBK, BMEC, LT, and Vero cell lines, exhibited moderate adaptability in BHK cells, and barely replicated in PK, F81, and 293T cell lines. Some of these findings are generally consistent with previous reports (Figure 4B) [38,43,44]. Obviously, although the above preliminary characterization of the ORF133 gene-deleted strain is extremely important, it is limited in scope. The deletion of the ORF133 gene may potentially cause side effects such as compensatory mutations, phenotypic changes, abnormal activation/inhibition of the host immune response, and alterations in host range and tissue tropism. In addition, the singularity of the in vitro experimental system and the lack of in vivo experiments fail to simulate the infection characteristics of the virus in different tissues inside and outside the body, which still warrants further investigation.

5. Conclusions

Bioinformatics analysis in this study revealed that the ORF133 gene is a conserved and unique gene in LSDV, which has a large genome. It has no clearly matched homologs in other poxvirus genera, and its function remains unknown, potentially encoding factors related to host range and virulence. To investigate the function of the ORF133 gene, a mouse-derived ORF133 polyclonal antibody with strong immunogenicity and high specificity was successfully prepared. Additionally, the ORF133 gene-deleted strain, LSDV-∆ORF133-EGFP, was constructed using homologous recombination technology. In vitro biological characteristic studies showed that although the ORF133 gene is not essential for LSDV replication, its deletion results in reduced replication kinetics of the virus in in vitro culture, suggesting a potential role in viral proliferation. In conclusion, the above results provide important experimental materials and technical theoretical support for further in-depth research on the biological function of the ORF133 gene and the pathogenic mechanism of LSDV.