Orf virus (ORFV) is the causative agent of orf, a contagious viral disease affecting various animal species, including goats and occasionally sheep, with zoonotic potential.1 ORFV is the prototype member of the genus Parapoxvirus, subfamily Chordopoxvirinae, family Poxviridae, along with bovine papular stomatitis virus, pseudoxinia virus, and New Zealand red deer paracinia virus.2 The disease primarily spreads through contact and exhibits epithelial tropism.3,4 Understanding the genetic characteristics of prevalent ORFV strains is paramount, not only for devising effective prevention and control measures but also for elucidating pathogenic mechanisms and developing intervention strategies.5–7
Research efforts worldwide have focused on dissecting the genetic diversity of ORFV, with particular emphasis on key genes like B2L, F1L, VIR, and VEGF.8,9 The F1L gene is the 59th open reading frame of the ORFV genome, located in a highly conserved region in the middle of the gene, encoding the resulting 39-kDa protein that is part of the microtubules on the viral surface and inducing the host to produce neutralizing antibody, and this protein has heparin-binding activity and can bind to the heparan sulfate receptor expressed on the surface of most mammalian cells, suggesting that this protein plays an important role in the viral adsorption and invasion process, and this protein is also an important component of ORFV capsule protein. It is 1 of the main antigen proteins that stimulate the body to produce an immune response and can mediate cellular immunity and humoral immunity.10,11 The B2L protein plays a key role in modulating cellular immunity and triggering a robust immune response in the host during the later stages of viral infection. It achieves this by encoding an immunogenic protein that forms part of the virus’ extracellular envelope. Additionally, the B2L protein exhibits lipase activity, which can increase the virus’ virulence. It also suppresses the host’s immune response, facilitating viral survival and replication within host cells.10,11 Therefore, it is important to study F1L and B2L protein for the diagnosis and immunity of ORFV.
Orf outbreaks have been documented in several regions of China, including Shandong, Guangxi, Shaanxi, Xinjiang, Henan, Heilongjiang, Yunnan, Anhui, Hainan, Fujian, and Jilin, causing significant economic losses to sheep and goat farmers.12,13 Therefore, understanding the genetic diversity of circulating ORFV strains in these regions is essential for devising targeted control measures and mitigating economic losses.
In the context of the Fujian province, China, where ORF outbreaks occur, a study conducted genetic analysis on 3 clinical samples from 2024, focusing on the cloning and analysis of the F1L and B2L genes. This endeavor aims to provide a scientific basis for effective orf prevention and control measures in the Fujian province, contributing to the broader understanding and management of ORFV infections.
Methods
Sample handling
In a biosafety cabinet, each lip scab skin was cut with scissors and ground in PBS at a volume ratio of 1:5. The resulting homogenate was then frozen at −20 °C. Subsequently, after centrifugation at 5,000 X g for 15 minutes, the supernatant was collected.
Primers design
Specific primers were designed with reference to the F1L and B2L gene sequences of the NZ2 strain (accession No. DQ184476.1) available in GenBank. The primer sequences are detailed in Supplementary Table S1.
ORFV F1L and B2L gene amplification
For amplification of the ORFV F1L and B2L genes, a reaction mixture consisting of 10 μL of High-Fidelity Enzyme Master Mix, 1 μL each of the upstream and downstream primers (10 μmol/L) targeting the F1L and B2L genes, 2 μL of DNA template, and RNase-Free H2O to make up a total volume of 20 μL was prepared. The reaction proceeded with an initial predenaturation step at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at the temperature specified in Supplementary Table S1 for 30 seconds, extension at 72 °C for 15 seconds, and a final extension step at 72 °C for 7 minutes by use of T100™ thermal cycler Bio-Rad Laboratories, Inc.
Sequence cloning of the ORFV strains F1L and B2L genes
The amplified target fragments were ligated into the cloning vector and transformed into Escherichia coli DH5α competent cells. Positive clones were identified, and the correct clones were selected for sequencing.
Sequence analysis of the ORFV F1L and B2L genes
Following sequence assembly, the nucleotide and deduced amino acid sequences of the ORFV F1L and B2L genes were compared with sequences from 22 ORFV strains and the B2L gene sequence of a Chinese vaccine strain (referenced in Supplementary Table S2). Subsequently, a genetic evolution analysis tree was constructed based on these sequences.
Statistical analysis
Homology analysis of the F1L and B2L genes was performed using DNASTAR MegAlign software. Wild and vaccine strains were used as the reference strains to analyze the variation in the F1L and B2L protein antigenic sites using DNASTAR Protein software. Using MEGA software, version 11, a genetic evolution tree was constructed for F1L and B2L by applying the neighbor-joining method.
Results
Results of PCR amplification of the ORFV F1L and B2L genes
In 3 clinical samples and ORFV China vaccine, bands of approximately 1,000 bp were specifically amplified, as depicted in Figure 1, aligning with the expected size. These samples were denoted as FJ-2401, FJ-2402, and FJ-2403 based on location and their collection time, respectively.
A—Results of PCR amplification of the F1L gene. M: DL2000 DNA marker. 1: China vaccine PCR product of the F1L gene. 2–4: 3 samples of the PCR product of the F1L gene. B—Results of PCR amplification of the B2L gene. M: DL2000 DNA marker. 1: China vaccine PCR product of the B2L gene; 2–4: 3 samples of the PCR product of the B2L gene.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.08.0222
Analysis of the nucleotide, deduced amino acid sequence similarity of the F1L gene
The sequences of the F1L genes for FJ-2401, FJ-2402, and FJ-2403 have been submitted to GenBank with accession numbers PP805862, PP805863, and PP805864, respectively. The alignment analysis revealed that the F1L gene in all 3 ORFV strains comprised 1,029 nucleotides, encoding 343 amino acids. The nucleotide sequence similarity of the F1L gene among the 3 ORFV strains was 98.6%, whereas the similarity in the deduced amino acid sequences ranged from 98.2% to 99.1%. Compared to domestic strains (NP, YX, GO, SJ1, CL18, SY17, NA17, GZ18, OV-HN3/12, and NA 1/11), the nucleotide sequence similarity ranged from 97.0% to 99.6%, and the similarity in deduced amino acid sequences ranged from 97.3% to 99.7%. Similarly, compared to foreign strains (ARA, CHB, HRE, MP, OV-SA00, OV-IA82, NAV, and UPM/HSN-20), the nucleotide sequence similarity ranged from 97.0% to 98.6%, and the similarity in deduced amino acid sequences ranged from 95.0% to 99.1%. For the weak strains (D1701 and Mukteswar_vaccine_passage50), the nucleotide sequence similarity was 96.3% to 98.3%, and the similarity in deduced amino acid sequences was 95.3% to 99.1%. Regarding the NZ2 reference strains, the nucleotide sequence similarity was 96.3% to 97.0%, whereas the similarity in deduced amino acid sequences was 95.8% to 96.1%. The amino acid sequence similarity with nucleotides and their deduced from Chinese vaccine strains ranged from 95.7% to 96.4% and 95.2% to 95.8%, respectively. The antigenic epitope prediction of F1L protein from Chinese vaccine strains and Fujian wild strains showed the mutation and deletion of amino acids in the F1L protein and showed obvious differences in the antigenic epitopes (Figure 2).
A—The prediction of the F1L protein antigenic index. B—Genetic evolutionary tree analysis of the F1L gene. All the names of the virus strains are listed in Supplementary Table S2.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.08.0222
Genetic evolutionary tree analysis of the F1L gene
The genetic evolution tree showed that the FJ-2401 and FJ-2402 strains were grouped together on the same evolutionary branch as the Fujian strains (NP, YX, and GO). Additionally, the FJ-2403 strain clustered with the GZ18 strain from Guangzhou, while the MP and Mukteswar_vaccine_passage50 strains from India were on the same evolutionary branch. They were distant from to NZ2 reference strain, Germany D1701 weak strain, and the China vaccine stain, which were located on different evolutionary branches (Figure 2).
Analysis of the nucleotide and deduced amino acid sequence similarity of the B2L gene
The sequences of the B2L genes for FJ-2401, FJ-2402, and FJ-2403 have been submitted to GenBank with accession numbers PP805859, PP805860, and PP805861, respectively. The alignment analysis revealed that the B2L gene consisted of 1,137 nucleotides, encoding 379 amino acids. The nucleotide sequence similarity of the B2L gene among the 3 ORFV strains ranged from 98.3% to 98.6%, whereas the similarity in deduced amino acid sequences ranged from 98.1% to 98.4%. Compared to domestic strains (NP, YX, GO, SJ1, CL18, SY17, NA17, GZ18, OV-HN3/12, and NA 1/11), the nucleotide sequence similarity ranged from 97.4% to 99.2%, and the similarity in deduced amino acid sequences ranged from 97.9% to 99.7%. Similarly, compared to foreign strains (ARA, CHB, HRE, MP, OV-SA00, OV-IA82, NAV, and UPM/HSN-20), the nucleotide sequence similarity ranged from 97.4% to 99.6%, and the similarity in deduced amino acid sequences ranged from 97.1% to 100%. For the weak strains (D1701 and Mukteswar_vaccine_passage50), the nucleotide sequence similarity was 97.9% to 98.2%, and the similarity in deduced amino acid sequences was 97.9% to 99.2%. Regarding the NZ2 reference strain, the nucleotide sequence similarity was 97.4% to 98.0%, whereas the similarity in deduced amino acid sequences was 97.1% to 98.4%. For the Chinese vaccine strains, the nucleotide sequence similarity was 97.7% to 98.2%, and the similarity in deduced amino acid sequences was 97.9% to 98.1%. The antigenic epitope prediction of B2L protein of Chinese vaccine strain and Fujian wild strain showed amino acid mutations in B2L protein and differences in antigenic epitopes (Figure 3).
A—The prediction of the B2L protein antigenic index. B—Genetic evolutionary tree analysis of the B2L gene.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.08.0222
Genetic evolutionary tree analysis of the B2L gene
The genetic evolution tree results revealed that the B2L gene exhibited 3 distinct branches in the 3 ORFV strains. Among them, FJ-2401 clustered with the Jilin (NA17 and CL18) strains on the same evolutionary branch. FJ-2403 grouped with the Fujian SJ1 strain and the Indian (Mukteswar_vaccine_passage50 and Mukteswar_passage9) strains within the same evolutionary clade. FJ-2402 clustered with the SY17 strain of Jilin, the MP strain of India, the UPM/HSN-20 strain of Malaysia, and the ARA strain of Argentina on the same evolutionary branch. However, the B2L gene in the 3 ORFV strains occupied different branches and were distantly related to the German D1701 weak strain, Fujian GO strain, and Chinese vaccine strain (Figure 3).
Discussion
Orf is generally overlooked worldwide due to its low incidence in sheep and goats. Early research data indicate that the incidence of this disease in the Fujian province ranges from 10.0% to 72.0%, with a mortality rate of 10.5% to 20.0%.12 In recent years, the incidence and mortality rate of orf have remained relatively low, failing to garner the attention of breeding enterprises in the Fujian province. However, with the zoonotic risk of the disease and recent reports of human infection,12,13 analyzing ORFV is crucial for scientifically preventing and controlling the disease in the Fujian province.
Previous studies6,9,10,14 have identified the B2L and F1L genes of ORFV as being located in highly conserved regions of the genome and typically serving as preferred genes for detecting and constructing genetic evolutionary trees. In this study, the B2L and F1L genes of 3 isolated ORFV strains were cloned and sequenced. The obtained sequences were uploaded to GenBank, and corresponding accession numbers were obtained. Concurrently, we compared the whole genomes of 22 ORFV strains included in the current GenBank and constructed a genetic evolution tree. The results showed that the nucleotide sequence similarity of the F1L and B2L genes in the 3 ORFV strains was 98.6% and 98.3% to 98.6%, respectively. The nucleotide sequence similarity with domestic epidemic strains was 97.0% to 99.6% and 97.4% to 99.2%, respectively, whereas with foreign popular strains it ranged from 97.0% to 98.6% and 97.4% to 99.6%, respectively. The nucleotide sequence similarity with weak strains (D1701 and Mukteswar_vaccine_passage50) and with the NZ2 reference strain was 96.3% to 98.3%, 97.9% to 98.2%, 96.3% to 97.0%, and 97.4% to 98.0%, respectively. The nucleotide sequence similarity with Chinese vaccine strain ranged from 95.7% to 96.4% and from 97.7% to 98.2%, respectively.
The genetic evolution tree of the F1L gene showed that the FJ-2401 and FJ-2402 strains grouped together on the same evolutionary branch as the Fujian strains (NP, YX, and GO). The FJ-2403 strain clustered with the GZ18 strain from Guangzhou, while the MP and Mukteswar_vaccine_passage50 strains from India on the same evolutionary branch. These strains were distant from the NZ2 reference strain, the Germany D1701 weak strain, and the China vaccine stain, which were located on different branches. Additionally, the genetic evolution tree results revealed that the B2L gene exhibited three distinct branches among the three ORFV strains. Specifically, the FJ-2401 strain grouped with the Jilin strains (NA17 and CL18) on the same evolutionary branch. The FJ-2403 strain clustered with the Fujian SJ1 strain and the Indian strains (Mukteswar_vaccine_passage50 and Mukteswar_passage9) within the same evolutionary clade. The FJ-2402 strain grouped with the SY17 strain from Jilin, the MP strain from India, the UPM/HSN-20 strain from Malaysia, and the ARA strain from Argentina on the same evolutionary branch. However, the B2L gene in these three ORFV strains occupied different branches and were distantly related to the German D1701 weak strain, the Fujian GO strain, and the Chinese vaccine strain. Antigenic epitope prediction of the F1L protein from Chinese vaccine strains and Fujian wild strains showed mutations and deletions of amino acids in the F1L protein, resulting in obvious differences in the antigenic epitopes. Similarly, antigenic epitope prediction of the B2L protein from the Chinese vaccine strain and Fujian wild strain revealed amino acid mutations in the B2L protein and differences in antigenic epitopes.
The above results suggest that the epidemic situation of ORFV in some sheep and goat farms in Fujian province is complex, with diverse ancestral sources, including domestic strains and foreign strains from India (MP, Mukteswar_vaccine_passage50, and Mukteswar_passage9). This indirectly implies that the current vaccine may not effectively protect against orf, thereby increasing the pressure on prevention and control efforts.
Wang et al15 analyzed the genetic characteristics of ORFV F1L and B2L genes in the Anhui province in 2018, finding that the Anhui ORFV epidemic strain was a new strain formed by genetic recombination of Shandong and Nantou epidemic strains. Ahanger et al16 analyzed the genetic characteristics of ORFV B2L and VIR genes in Kashmiri, showing that the locally circulating ORFV sheep isolate had high homology with Greece and Italy, whereas the ORFV goat isolate had high homology with Chinese goat isolates. The conclusions of these researchers are consistent with this study, indicating that future circulating strains of ORFV will mainly be genetic recombinant strains. Analyzing ORFV virulence-related genes is beneficial for understanding differences in pathogenicity among different ORFV strains, and discovering related virulence genes can provide an important theoretical basis for constructing ORFV gene deletion vaccines and preparing virulent vaccines.
This study obtained B2L and F1L gene sequences from 3 ORFV strains, enriching the database of ORFV-related gene sequences. It clarified the current molecular epidemic characteristics of ORFV in some sheep farms in the Fujian province and provided a theoretical basis for the effective prevention and control of orf in the Fujian province in the future.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
This work was supported by the Fujian Province Agricultural High-Quality Development beyond “5511” Collaborative Innovation Project (grant No. XTCXGC2021008), Fujian Public Welfare Project (grant Nos. 2020R1026009 and 2021R1026007), Natural Science Foundation of Fujian Province (grant No. 2021J01484), Science and Technology Innovation Team Building Project of Fujian Academy of Agricultural Sciences (grant No. CXTD2021007-2), and Fujian Province 2022 Central Leading Local Science and Technology Development Fund (grant No. 2022L3085).
ORCID
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