2.1. Cells and Reagents

Madin–Darby bovine kidney (MDBK) cells were obtained from the American Type Culture Collection (ATCC CCL-22). The cells were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and penicillin (100 IU/mL) and streptomycin (10 μg/mL) at 37°C and 5% CO₂. Specific pathogen-free (SPF) 9-day-old chicken embryos were provided by Boehringer Ingelheim Viton (Beijing, China). Mouse polyclonal antibodies against the LSDV A27 protein were prepared in our laboratory.

2.2. Sample Collection and Processing

From a cattle farm in Shaanxi Province, Northwest China, the skin and tracheal mucosal nodule contents of dead cattle suspected of having LSD were collected aseptically using scalpels and tweezers, temporarily stored in sterile test tubes, and transported to a designated laboratory at a low temperature throughout the process. Using sterile scissors and tweezers, 0.2 g of diseased tissue were removed, and five times the volume of precooled PBS was added to disperse the tissue using a tissue grinder. The posttreatment samples were thrice frozen and thawed at −80°C to release the virus in the cells. Then, the supernatant was collected by centrifugation at 5000 rpm for 10 min and used for qPCR detection and virus isolation. Part of the supernatant was used for qPCR detection, and the other mixed 1:1 with a penicillin–streptomycin-mixed solution (100x) and incubated overnight at 4°C to completed the pretreatment before virus isolation.

2.3. qPCR Detection and Virus Isolation

For qPCR detection, the EasyPure viral DNA/RNA kit (TransGen Biotech, Beijing, China) was used to extract viral DNA from the collected supernatant, and the eluted viral DNA was stored at −20°C. Fluorescent quantitative primers and probe were designed in the LSDV ORF 007 region for qualitative sample detection (Table 1). Primers and probe were synthesized by Tsingke Biotechnology (Beijing, China). The HS Premix Probe qPCR Kit III was purchased from Accurate Biotechnology (Hunan, China). A two-step qPCR amplification procedure was used as follows: 95°C for 30 s followed 45 cycles of 95°C for 5s and 60°C for 30 s. Target gene amplification and signal acquisition were performed using a Bio-Rad CFX Connect (California, America) instrument.

MDBK cells were cultured to 80% confluence, incubated with supernatant treated with penicillin–streptomycin solution for 2 h. The supernatant was discarded and DMEM supplemented with 2% FBS was added to continue the incubation to wait for the appearance of obvious cytopathic effect (CPE). If no obvious CPE was observed, the cell samples were collected by repeated freeze-thawing for the next passaging before the cell state deteriorated, and the samples were passed through three to four generations until the CPE was stable. After 80% of the cells showed CPE, they were thrice frozen and thawed, centrifuged at 5000 rpm for 10 min to collect the supernatant, and refrigerated at −80°C for later use.

For the chicken embryo isolation virus, the treated samples were inoculated with 9-day-old SPF chicken embryos through a chorioallantoic membrane and cultured in an incubator at 38°C for 5 days. Chicken embryos that died within 24 h after inoculation were discarded, and the embryos were dissected after 5 days to observe any chorioallantoic membrane lesions. The parts with thickened chorioallantoic membranes and pockmarks were collected. After suspension in PBS, the supernatant was thrice frozen and thawed and centrifuged at 5000 rpm for 10 min. The collected samples were passaged three times in succession on new SPF chicken embryos.

2.4. Indirect Immunofluorescence Test

To determine whether the isolated virus bound to LSDV-specific antibodies, LSDV-infected MDBK cells were immobilized with 4% paraformaldehyde for 30 min and then infiltrated with 0.2% Triton X-100 for 20 min. A mouse polyclonal antibody against the LSDV A27 protein was used as the primary antibody at a dilution ratio of 1:500; FITC-labeled goat anti-mouse IgG (H + L; Biodragon, Jiangsu, China) was used as the secondary antibody, diluted at 1:1000; and DAPI solution (Solarbio, Beijing, China) was used to label the nucleus and perform an indirect immunofluorescence assay. The stained cells were observed, and images were collected using a Zeiss inverted fluorescence microscope.

2.5. Transmission Electron Microscope

MDBK cells with 80% confluence were inoculated with LSDV (MOI = 5) and incubated for 2 h. After replacement of the maintenance medium, the cells were cultured until notable CPE occurs. Trypsin was used to digest dispersed cells and 3% glutaraldehyde to fix the cells. Finally, JEM-1400FLASH transmission electron microscope (Japan Electronics Corporation) was used for image acquisition.

2.6. Virus Biological Characteristics

Virus titers of the LSDV/China/SX/2023 isolates were determined using the TCID₅₀ assay and calculated using the Reed–Muench method. The LSDV growth curve was measured in MDBK cells which were inoculated at one MOI infection dose, and samples were collected every 24 h after inoculation for 9 days. Finally, viral titers at each time point were measured using TCID₅₀.

2.7. Whole-Genome Sequencing Analysis

First, high quality viral DNA was prepared using the EasyPure viral DNA/RNA kit, then sequencing libraries were constructed using Covaris M220 fragment viral genomic DNA and the TruSeq DNA sample prep kit. Sequencing was performed according to the Illumina NovaSeq 6000 protocol, and the data were sorted and analyzed. SOAPdenovo (version 2.04) was used for the genome splicing and assembly, and the assembled viral genome was named LSDV/China/SX/2023.

2.8. Phylogenetic and Gene Recombination Analyses

Using the CaPV whole genome sequence in the NCBI database as a reference (Table 2), Mafft was used to perform multiple sequence alignments of 38 CaPV genome sequences, including LSDV/China/SX/2023. Trim and align sequences were performed with the multiple alignment trimming plugin in Tbtools. The maximum likelihood method in Mega 11 was used to construct an evolutionary tree to characterize the evolutionary and kinship relationships between LSDV/China/SX/2023 and the previously reported CaPVs (BOOTSTRAP value set to 1000). Itol (https://itol.embl.de/itol.cgi) was used to construct the evolutionary tree.

Based on the CaPV genome alignment sequence, the viral recombination event was analyzed using the RDP4 software and seven methods: RDP, GENECONV, Chimera, MaxChi, BootScan, SiScan, and 3Seq. Based on RDP4 analysis, recombination events were further verified and characterized using Simplot.

3.1. Virus Isolation and Culture Characteristics

qPCR primers and probes targeting the 007 ORF region of the LSDV genome were used for qualitative detection of LSDV. The CT value of the tested samples was 18 and a negative control was established (Figure 1). After antibiotic treatment, the sample supernatant with positive qPCR results was inoculated into MDBK cells. CPE appeared in the cells after two generations of blind transmission, manifesting as local cells aggregation, and surrounding cells were deformed and elongated (Figure 2). The chorioallantoic membranes of SPF chicken embryos inoculated with the positive samples showed notable mucosal thickening and specific acne-like lesions (Figure 3).

3.2. Indirect Immunofluorescence Identification

To determine whether local cell lesions were caused by LSDV-specific infection, an indirect immunofluorescence assay was performed using a preprepared anti-LSDV A27 protein mouse polyclonal antibody. Under a fluorescence electron microscope, specific green fluorescence was clearly observed at the site of clustered cytopathic lesions, and no green fluorescence was observed in the non-CPE and uninfected groups (Figure 4).

3.3. Observation by Transmission Electron Microscope

Poxvirus virions are generally >200 nm in diameter; most of them are brick- or oval-shaped and have two forms: extracellular enveloped virions (EEVs) and intracellular mature virions (IMVs) [27]. The LSDV-infected cells were fixed with glutaraldehyde and observed under a transmission electron microscope. Many uniform particle size virions in the range of 200–250 nm were observed. The particles were either brick or oval shaped. In the same field of view, two viral morphologies were observed, EEVs and IMVs, in which EEVs clearly had a double envelope morphology (Figure 5).

3.4. Virus Biological Characteristics

The TCID₅₀ of LSDV used to perform the virus biological characteristics experiment was measured to be 10^−5.8/mL. After inoculation of MDBK cells with LSDV at one MOI infection volume, the viral titer gradually increased from the first day after inoculation, peaked on the 7th, and began to slowly decline thereafter (Figure 6).

3.5. Genome Sequence Comparison Between LSDV Isolates and Reference Strains

We uploaded the whole genome sequence of the isolated LSDV strain to NCBI database and named it LSDV/China/SX/2023 (GenBank: PP894832). The nucleotide identities of the LSDV/China/SX/2023 and LSDV strains isolated in China were >99.9%. The nucleotide homogeneity of LSDV/China/SX/2023 with GTPV and SPPV was <98.0%, whereas that of LSDV/China/SX/2023 with GTPV AV41 was only 97.6% (Table 3).

A sequence comparison revealed that LSDV/China/SX/2023 had base insertions or mutations at ORFs 19, 59, and 143 compared with LSDV/China/XJ01/2019, which directly led to changes in the amino acid homology of the Kelch-like protein (ORF 19), poxvirus myristoyl protein (ORF 59), and tyrosine protein kinase-like protein (ORF 143) with altered amino acid homology (Table 4). Compared with LSDV/China/GD01/2020, LSDV/China/SX/2023 had five ORFs coding for products with <100% identity, namely, the hypothetical protein (ORF 1), Kelch-like protein (ORF 19, ORF 144), putative ER-localized apoptosis regulator (LSDV154), and hypothetical protein (LSDV155; Table 4). LSDV/China/SX/2023 showed no correlation between the base insertions or mutations in these ORFs and their corresponding positions in GTPV and SPPV.

Notably, the LSDV/China/SX/2023 viral genome contains a base A insertion at 13229. This base insertion causes the length of ORF 19 change from 1323 to 873 bp, and the molecular weight of the Kelch-like protein encoded by it change from 51.4 to 33.5 kDa. In addition, the base insertion was specific to the whole genome sequence of 37 CaPV strains worldwide and was not found in either vaccine or field strains (Figure 7).

3.6. Evolutionary Tree Analysis of LSDV Isolates

Phylogenetic trees constructed using maximum likelihood suggest that LSDV/China/SX/2023 is in the same clade as the LSDV recombination strains isolated from China and Southeast Asia after 2019, as well as the two LSDV strains isolated from Russia after 2020. LSDV phylogeny can be divided into three main branches of vaccine, field, and vaccine-recombinant strains, and there are three secondary branches of vaccine-recombinant strains, the most important of which is composed of the Asian-recombinant strains (Figure 8). SPPV and GTPV are two separate evolutionary clades genetically closer to the LSDV field strains.

3.7. Virus Recombination Event Prediction

To identify recombination events in the LSDV/China/SX/2023 strain, seven methods in RDP4 were used to predict the viral recombination events and sites. The prediction results showed that there were 15 recombination events in LSDV/China/SX/2023 with each event supported by at least two methods (p  < 0.1). LSDV/China/SX/2023 had the same recombination event as LSDV strains isolated from China and Southeast Asia after 2019. To predict recombination event of CaPV, we jointly analyzed the genomic data for GTPV. Notably, 17 recombinant strains from Asia had a GTPV gene recombination event in the breakpoint region (135621–138634), one of the 15 recombination events. The Simplot software and RDP4 results corroborated (Figure 9).