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Recombinant A27L Protein and Real-time PCR-based Detection of Lumpy Skin Disease Virus: A Step Toward Enhanced Diagnosis and Vaccine Research

P
Pravalika Annapureddy1
V
Vandana Gupta1,*
G
Gulshan Kumar1
M
Megha Katare Pandey2
A
Anju Nayak1
S
Swati Tripathi1
R
Renuka Mewade1
S
Sai Venkatesh Yakkali3
1Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
2Department of Translational Medicine, All India Institute of Medical Sciences Bhopal, Bhopal-462 026, Madhya Pradesh, India.
3Department of Veterinary Pathology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.

ABSTRACT

Background: Lumpy skin disease (LSD) presents a significant threat to the global livestock industry, with potential implications for both animal health and economic stability. The recombinant A27L protein, derived from the lumpy skin disease Virus (LSDV), is a candidate for both diagnostic applications and vaccine development.

Methods: In this study, we successfully cloned, expressed and purified the A27L protein using an E. coli expression system. A 447 bp fragment of the A27L gene was amplified from LSDV DNA, cloned into the pJET1.2/blunt vector and subcloned into the pQE30 expression vector. Expression of the recombinant protein was induced in E. coli M15 cells, yielding a 17.7 kDa protein, which was subsequently purified using nickel affinity chromatography. Additionally, a real-time PCR assay was performed for the differentiation of LSDV from other related poxviruses, enhancing the specificity of diagnostic testing.

Result: Preliminary diagnostic validation of A27L protein was conducted using a DOT ELISA with LSDV-positive field serum sample, showing promising results. While the initial findings suggest the potential of the recombinant A27L protein for diagnostic use, further validation, including larger sample sizes and comparisons with existing diagnostic methods, is necessary to fully establish its effectiveness as a diagnostic tool and vaccine candidate. In addition, real-time PCR assay demonstrated specific detection and differentiation of LSDV from other related poxviruses.

INTRODUCTION

Lumpy skin disease (LSD), also known as Neethling virus disease, is a re-emerging, vector-borne disease of cattle, causing significant economic losses globally. Initially confined to sub-Saharan Africa, it has now become transboundary, with outbreaks reported in the Middle East, Europe, Russia and Asia (Babiuk et al., 2008). LSD is characterized by high fever (>104oF), multiple skin nodules, mucosal lesions, weight loss, lymphadenopathy, brisket edema and complications such as reduced milk yield, abortions, infertility, hide damage and secondary infections (Bedekovic et al., 2018). The disease is caused by the Neethling strain of Lumpy Skin Disease Virus (LSDV), a member of the genus Capripoxvirus, subfamily Chordo-poxvirinae, family Poxviridae (King et al., 2012). Transmission occurs via direct contact with infected secretions (nasal discharge, saliva, blood, milk, semen), intrauterine transmission and primarily through insect vectors like mosquitoes, ticks, flies and midges (Gupta et al., 2020; Sprygin et al., 2019). LSD typically shows high morbidity (up to 50%) and low mortality (<10%), though these rates vary with host immunity and vector prevalence (Das et al., 2021; Manić et al., 2019).
       
LSDV is an enveloped virus with a dumbbell-shaped core and lateral bodies. Its 151 kbp double-stranded DNA genome includes 2.4 kbp inverted terminal repeats and encodes 156 ORFs, of which 146 are conserved and essential for replication, transcription and assembly (Tulman et al., 2001). Its 97% genomic similarity with Goatpox (GTV) and Sheeppox viruses (SPV) complicates differential serological diagnosis (Tulman et al., 2001; Bianchini et al., 2023). LSDV ORF 117 is an ortholog of the Vaccinia virus (VV) A27L protein, which is found on the surface of the intracellular mature virion (IMV) and plays a key role in virus-cell binding, membrane fusion and intracellular transport (Chung et al., 1998; Blouch et al., 2005). A27L is essential for viral morphogenesis, assembly and release, interacting with other viral proteins such as A17L to maintain structural integrity and infectivity (Kumar et al., 2015; Hsiao et al., 1998). The protein features a coiled-coil helical region that forms trimers, crucial for its functional activity (Rodriguez et al., 1987; Sodeik et al., 1995). Due to its role in immune evasion and infection, A27L has been identified as a potential antigen and target for neutralizing antibodies in poxviruses, including sheeppox and vaccinia virus (Chervyakova et al., 2016). It has demonstrated the ability to induce virus-neutralizing antibodies in mice and provides protective immunity when used as a vaccine target (Demkowicz et al., 1992; Berhanu et al., 2008; Rudraraju and Ramsay 2010). Monoclonal antibodies against A27L can neutralize viral infectivity, offering passive protection (Ramírez et al., 2002). Additionally, recombinant A27L proteins from various poxviruses are used for diagnostic purposes, including sero- monitoring and surveillance during outbreaks (Dashprakash et al., 2015; Chervyakova et al., 2016). In addition to protein-based diagnostics, Real-time PCR (qPCR) has become an invaluable tool for detecting and quantifying LSDV. Compared to conventional PCR, qPCR offers superior sensitivity, speed and reproducibility, making it an essential method for rapid diagnosis and timely intervention during outbreaks. By utilizing universal capripox primers and species-specific probes targeting conserved genetic regions, such as the RPO30 gene, qPCR allows for the differentiation of LSDV from other capripox viruses like Goatpox and Sheeppox (Wang et al., 2021). The use of qPCR in diagnostic protocols is vital for monitoring vaccine efficacy, controlling the spread of LSDV and conducting epidemiological studies, especially as capripox viruses continue to spread globally.
       
This study aims to clone, express and purify the recom-binant A27L protein from LSDV and enhance diagnostic capabilities through differentiating real-time PCR. The combination of these two approaches holds promise for advancing the understanding and control of LSD, paving the way for better diagnostic tools and more effective vaccine development.

MATERIALS AND METHODS

Place of work and sample collection
 
This study was carried out at the Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur, Madhya Pradesh from 2022-2024. Among the LSDV-positive tissue samples present in the department, one was selected for downstream cloning and expression of the target gene. In total, eleven LSDV tissue samples were screened using a real-time PCR assay for the detection of lumpy skin disease virus (LSDV). Subsequently, these samples were subjected to differential diagnosis to distinguish LSDV from goatpox virus.

Genomic DNA extraction
 
Genomic DNA from tissue samples and goat pox vaccine (Hester Biosciences Limited) was extracted using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s protocol.
 
Gene amplification
 
The extracted LSDV DNA was then subjected to PCR amplification using the GeneAmp PCR System 9700 (Applied Biosystems). The A27L gene fragment was amplified from the genomic DNA using the forward primer -AAT GGA TCC ATG GAC AGA GCT TTA TCA ATC TTT C and reverse primer R-AAT GTC GAC TCA TAG TGT TGT ACT TCG GCC, as described by (Ntombela et al., 2023) under the following PCR conditions: Initial denaturation at 95oC for 5 minutes, followed by 35 cycles consisting of denaturation at 95oC for 1 minute, annealing at 60°C for 40 seconds, extension at 72oC for 1 minute and a final elongation step at 72oC for 7 minutes. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel, using a 100 bp DNA ladder as a size marker.
 
Cloning of A27 l gene
 
The amplified PCR product for A27L gene was purified using Qiagen QIAquick® gel extraction kit (Qiagen, Germany) according to the manufacturer’s protocol. The cloning of the A27L gene using the pJET1.2/blunt vector was carried out as per the protocol provided by the CloneJET PCR Cloning Kit manufacturer. In order to insert the desired gene into the cloning vector, the purified PCR product with sticky ends needs to be converted into a blunt-ended form using DNA blunting enzyme. The blunt ended PCR product was ligated into the pJET1.2/blunt cloning vector using T4 DNA ligase enzyme. Following ligation, the recombinant plasmid, pJET1.2/blunt-A27L, was transformed into E. coli DH5α cells, which were selected on ampicillin-containing Luria Bertani (LB) agar plates. The resulting colonies were screened by colony PCR using both insert and vector primers (provided in the kit) to identify successful transformants and the PCR products were analyzed by agarose gel electrophoresis, confirming the presence of the 447 bp A27L fragment. To validate the cloning process, a positive control (provided in the kit) was utilized. Recombinant plasmid from the positive colonies was isolated using Favor PrepTM Plasmid Extraction Mini Kit (Favorgen, Taiwan). The recombinant plasmid (pJET1.2 and A27L) was digested with two restriction endonucleases, BamHI and Sal I. The digested PCR product was analysed by running it on a 1% agarose gel.
 
Expression of A27L fusion protein
 
The digested A27L gene and the pQE30 vector (BamHI and SalI restriction enzyme digested) fragments were purified using the Qiagen QIAquick® Gel Extraction Kit (Qiagen, Germany) according to the manufacturer’s protocol. Ligation of RE digested vector and insert was carried out using T4 DNA ligase. Recombinant plasmid vector pQE30-A27L was transformed into E. coli M15 cells and selected on ampicillin kanamycin containing LB agar plates. The colonies were screened for the presence of recombinant plasmid pQE30-A27L by colony PCR and also isolated recombinant plasmid was checked for insert release by double digestion with BamHI and Sal I restriction endonuclease followed by analysis of digested products on 0.8% agarose gel. For prokaryotic expression, recombinant bacterial clones containing the insert were cultured in 10 ml of LB broth supplemented with ampicillin (100 µg/ml) and kanamycin (50 µg/ml), with continuous shaking. Once the culture reached an optical density at 600 nm (OD 600) of 0.6 to 1.0, 1 mM IPTG was introduced to induce protein expression and the culture was incubated with constant shaking at room temperature. Two ml of the induced culture was collected every 2 hours starting from 2h onward up to 10 h. All the cultures collected were pelleted by centrifugation at 13,000 rpm and stored at -20oC for SDS-PAGE analysis.
       
In this step, a positive control plasmid was also utilized. The control expression plasmid (pQE40), which encodes a 26 kDa protein, served as a positive control for expression, as outlined in the QIAexpress® Type IV Kit from Qiagen (Germany). This positive control is essential for validating the expression system and ensuring the reliability of the results obtained.
 
Purification of expressed protein 
 
The recombinant protein with histidine residues at N-terminal end of the protein was purified under denaturing conditions using Ni-NTA agarose. Around 250 ml culture was induced with 1 mM IPTG for 12 hrs under constant shaking at room temperature. The culture was pelleted by centrifugation at 13,000 rpm for 20 minutes. The obtained bacterial pellet was resuspended in 10 ml lysis buffer (8 M urea, 0.1 M NaH2PO4, pH 8.0). Incubation was carried out at room temperature for 1 hr. by gently swirling the cell suspension. Lysis was complete when the suspension gets translucent. Lysate was centrifuged at 14,000 rpm for 30 min at room temperature to pellet the cellular debris. Cell lysate supernatant containing the recombinant protein was applied to polypropylene column filled with one ml of Ni-NTA agarose. Flow through fraction was collected. Column was washed 2 times with 4 ml of wash buffer (8 M urea, 0.1 M NaH2PO4, pH 6.3). Wash fractions were also collected. 6x His-tagged protein was eluted three times with 0.5 ml of elution buffer D (8 M urea, 0.1 M NaH2PO4, pH 5.9) and collected as 500 µl aliquots.  Second elution was done three times with 0.5 ml of elution buffer E (8 M urea, 0.1 M NaH2PO4, pH 4.5) and collected as 500 µl aliquots. Twenty microlitre of each sample was mixed with equal volume of 2X SDS-PAGE sample buffer and stored at -20oC for SDS-PAGE analysis. The purified protein was estimated by lowry method.
 
DOT ELISA for purified protein
 
A 10 µL drop of peptide solution, with a concentration of 108 ng/µL, was applied onto a nitrocellulose membrane and allowed to dry for 10 minutes at room temperature. A negative control with PBS (Phosphate buffered saline) was used. Following the drying step, the membrane underwent three rounds of washing, each lasting 2 minutes, using a 0.1% solution of PBS-T (Phosphate-buffered saline with 0.05% Tween-20). Subsequently, the membrane was subjected to a blocking step by incubating it at 37oC for 1.5 hours with a blocking solution containing 5% bovine serum albumin (BSA) and 0.05% PBS-T. After the blocking procedure, the membrane was washed three times. For primary antibody incubation, LSDV-positive field serum was applied as the primary antibody and incubated for 45 minutes at 37oC. Following three additional washes with PBS-T, a secondary antibody conjugated to horseradish peroxidase (HRP), specifically anti-bovine HRP, was applied at a dilution of 1:2000. This incubation was carried out for 40 minutes at 37oC. After the final three washes, color development was initiated by preparing a reaction mixture consisting of 6 mg of 3, 3’-diaminobenzidine (DAB) dissolved in 10 mL of PBS, to which 10 µL of hydrogen peroxide was added. The reaction was stopped by the addition of an excess amount of double-distilled water.
 
Real time PCR
 
Real-time PCR (qPCR) was conducted on eleven LSDV-positive tissue samples from the Department of Veterinary Microbiology, NDVSU, Jabalpur. Amplification was performed using universal Capripoxvirus primers and an LSDV-specific probe on the QuantStudio™ 5 Real-Time Detection System (Applied Biosystems, Thermo Fisher Scientific). Data were analyzed using QuantStudio™ Design and Analysis Software (version 1.3.1). The Neoscript One-Step qRT-PCR Kit (Genes2Me, India) was used for all reactions, supplying the necessary reagents for efficient amplification and detection, as per the manufacturer’s protocol.
       
The universal Capripoxvirus primers used for the real time amplification were -forward: 5′-ATG GTA GGA TAG TCG CAA ATG AT-3′  and reverse: 5′ -AGA TAT AAA CCC GGC AAG TGA C-3′ -along with species-specific probes: LSDV probe (5′ -FAM-TAA GCG ATT TTA TAG TTG CAA TGC GTA GT-BHQ-3′) and GTPV/SPPV probe (5′ -SUN-TAA GCG ATT TTA TAG TTG CGA TGC GTG GC-BkFQ-3′). Each 20 µl qPCR reaction contained 1.0 µl of each primer, 2.0 µl of template DNA, 1.0 µl of LSDV probe, 10.0 µl of master mix and 5.0 µl of nuclease-free water. Amplification was carried out in 0.2 ml PCR tubes using the following thermocycling conditions: initial denaturation at 95oC for 5 min, followed by 40 cycles of 95oC for 15 sec and 61oC for 35 sec (Wang et al., 2021).
       
To differentiate LSDV from related Capripoxviruses (GTPV/SPPV), the assay was further optimized by testing each viral DNA template with both probes. No cross-reactivity was observed, confirming probe specificity. Duplex qPCR assays were then developed using both probes and both templates in a single 25 µl reaction (12.5 µl master mix, 0.5 µl enzyme, 2.0 µl each primer, 1.0 µl each probe, 1.5 µl each template and 3.0 µl nuclease-free water). This setup enabled simultaneous detection and differentiation of LSDV and GTPV/SPPV in a single assay, demonstrating high specificity and utility for field diagnostics.

RESULTS AND DISCUSSION

Amplification and cloning of LSDV A27L
 
The A27L gene fragment from LSDV was successfully amplified, yielding a 447 bp amplicon (Fig 1). The PCR product was purified from an agarose gel and subsequently made blunt ended to facilitate ligation into the pJET1.2/blunt cloning vector. The ligation mixture was then transformed into E. coli DH5α competent cells (Fig 2). Transformants were screened by colony PCR using both insert-specific and vector-specific primers. PCR products amplified using the insert primers were analyzed by 1% agarose gel electrophoresis, confirming the presence of the expected 447 bp A27L fragment in positive colonies.

Fig 1: Agarose gel electrophoresis showing amplified A27L Fusion gene.



Fig 2: Transformation of plasmid (pJET1.2 + A27L) recombinant into E. coli DH5α cells.


       
The PCR products using vector primers resulted a product size of 550 bp. In the process of amplifying a recombinant plasmid using vector-specific primers, the PCR amplifies not only the insert but also segments of the vector flanking the insertion site. Hence, the size of the amplicon was greater (550bp) than the size of the insert (447 bp) alone. Colony PCR was also performed for positive control (provided in the kit), resulting in the amplification of a DNA fragment with a size of 976 bp (Fig 3). This size is consistent with the specifications provided in the cloning kit manual (CloneJET PCR Cloning Kit), confirming the successful incorporation of the insert. After confirmation through colony PCR, recombinant plasmid (pJET1.2/blunt+ A27L) from the colonies was isolated and RE digested using BamHI and Sal I restriction endonucleases.

Fig 3: Colony PCR using vector primers after transformation in pJET1.2 vector.


 
Expression and purification of A27L fusion protein
 
The restriction enzyme (RE)-digested A27L gene was ligated into the similarly digested pQE30 expression vector to form the recombinant construct pQE30-A27L. The plasmid was transformed into E. coli M15 cells and plated onto LB agar containing ampicillin and kanamycin for dual antibiotic selection (Fig 4). Transformants were screened by colony PCR using A27L-specific primers and the positive clones provided the expected 447 bp amplicon (Fig 5), confirming the successful insertion of the A27L gene into the expression vector. This two-vector cloning approach was deliberately employed to ensure greater precision and efficiency in expression. While direct cloning into the pQE30 expression vector is feasible, the use of pJET1.2/blunt as an intermediate vector provided an additional layer of monitoring.

Fig 4: Growth of recombinant (pQE30 + insert) M15 cells in LB agar containing ampicillin and kanamycin.



Fig 5: Colony PCR amplification of A27L fusion gene visualized by agarose gel electrophoresis.


      
The positive recombinant colonies were induced with 1/ mM IPTG to initiate protein expression. While not fully induced, expression of the 17.7/ kDa A27L protein could be detected as early as 2 hours after induction, with band intensity progressively increasing up to 6 hours (Fig 6). The positive control (pQE40 plasmid) detected a clear band at approximately 26/ kDa, demonstrating that the positive control was functional expression system. Uninduced cells and control cultures did not exhibit visual additional bands, confirming with assurance that no leaky or non-specific expression was occurring. The cloning and expression of the A27L gene resulted in the production of a fusion protein with a size of 17.7 kDa. This finding is consistent with the work of (Ntombela et al., 2023), who reported an expressed protein of 21 kDa. The observed variation in protein size can be attributed to the different expression vectors utilized in the respective studies. In our research, we employed the pQE-30 vector, while (Ntombela et al., 2023), utilized the pET28a vector for cloning and expression. The differences in vector design and features can influence the processing and translation of the protein, leading to variations in the final protein size. The expressed recombinant A27L protein was purified from bacterial lysates with Ni-NTA metal chelate affinity chromatography. Following purification, a single sharp band at 17.7/ kDa that corresponded to A27L was clearly visualized on 12% SDS-PAGE, confirming confirmation that the target protein was successfully recovered and pure (Fig 7).

Fig 6: SDS PAGE before purification.



Fig 7: SDS PAGE after purification.


 
DOT ELISA
 
The immunogenicity of the purified recombinant protein was subsequently assessed using DOT ELISA. The expressed protein reacted with LSDV-positive field serum, producing a visible color change, while the negative control (PBS) showed no reaction (Fig 8). This demonstrates that the recombinant protein is specifically recognized by antibodies generated during LSDV infection, indicating that the A27L protein contains immunodominant epitopes. Immunoreactivity observed here aligns with the fundamental principle that recombinant viral proteins, when expressed correctly, can mimic native viral antigens and elicit specific antibody recognition. The absence of cross-reactivity with negative controls strengthens the reliability of this assay. These findings suggest that the recombinant A27L protein could serve as a candidate antigen for serological testing in diagnostic applications. However, broader validation with a larger panel of serum samples, including those from animals infected with other pathogens, will be essential to confirm specificity and rule out cross-reactivity.

Fig 8: DOT ELISA for purified recombinant fusion protein.


 
Real time PCR
 
Real-time PCR analysis of eleven LSDV-positive tissue samples using universal Capripoxvirus primers and LSDV-specific probes yielded cycle threshold (Ct) values ranging from 13.905 to 20.538 (Fig 9). These values are in agreement with (Singh et al., 2024), who reported mean Ct values of 23.94±2.97 for skin biopsy samples and 25.23±2.41 for intranodular saline lavage and with (Sanganagouda et al., 2023), who observed Ct values between 6.81 and 23.23 in skin swabs. The relatively lower Ct values observed in this study suggest a higher viral load, reflecting the high sensitivity of real-time PCR for LSDV detection. These findings support the utility of real-time PCR in timely diagnosis and outbreak monitoring. Our results also align with (Wang et al., 2021), who noted that LSDV Ct values typically fall below 30. Such consistency across studies reinforces the robustness of the assay across different sample types and viral loads, underscoring its value in both clinical diagnostics and epidemiological surveillance.

Fig 9: Amplification of Lumpy skin disease virus using real-time PCR, Ct values ranging from 13.905 to 20.538.


       
A single-reaction real-time PCR assay was developed using both LSDV and GTPV templates, universal Capripox primers in combination with species-specific probes for LSDV and SPPV/GTPV. This assay successfully differentiated LSDV from GTPV, with the SPPV/GTPV probe amplifying GTPV at a Ct value of 18.096, while the LSDV probe detected LSDV at a significantly lower Ct value of 11.531 (Fig 10).

Fig 10: Real time PCR using both probes and both templates, GTPV with a Ct value of 18.096 (Target 1), LSDV with a Ct value of 11.531 (Target 2).


       
These findings are consistent with (Wang et al., 2021) and (Nan et al., 2023), who demonstrated that the GTPV/SPPV probe specifically detects GTPV DNA without amplifying LSDV and vice versa for the LSDV probe. The results validate the specificity and diagnostic effectiveness of the real-time PCR assay for differentiating Capripoxvirus infections.

CONCLUSION

This study successfully demonstrated the cloning, expression, and purification of the recombinant A27L protein from Lumpy Skin Disease virus, with preliminary findings showing its immunogenicity through specific recognition by LSDV-positive field serum samples, indicating potential diagnostic utility. However, the current results are preliminary due to limited sample size and lack of statistical validation. Further studies involving larger sample sets, control groups, cross-reactivity testing, and animal model evaluations are required to confirm its diagnostic specificity, sensitivity, and vaccine potential. Additionally, the GTPV/SPPV and LSDV probes showed high specificity by exclusively detecting their respective viral DNAs without cross-reactivity, supporting their usefulness in differentiating LSDV, GTPV, and SPPV infections for accurate diagnostics.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

REFERENCES


  1. Babiuk, S., Bowden, T.R., Boyle, D.B., Wallace, D.B. and Kitching, R.P. (2008). Capripoxviruses: An emerging worldwide threat to sheep, goats and cattle. Transboundary and Emerging Diseases. 55(7): 263-272.  

  2. Bedekovic, T., Simic, I., Kresic, N. and Lojkic, I. (2018). Detection of lumpy skin disease virus in skin lesions, blood, nasal swabs and milk following preventive vaccination. Transboundary and Emerging Diseases. 65(2): 491-496. 

  3. Blouch, R.E., Byrd, C.M. and Hruby, D.E. (2005). Importance of disulphide bonds for vaccinia virus L1R protein function. Virology Journal. 2: 1-5.

  4. Berhanu, A., Wilson, R.L., Kirkwood-Watts, D.L., King, D.S., Warren, T.K., Lund, S.A., Brown, L.L., Krupkin, A,K., VanderMay, E., Weimers, W. and Honeychurch, K.M. (2008). Vaccination of BALB/c mice with Escherichia coliexpressed vaccinia virus proteins A27L, B5R and D8L protects mice from lethal vaccinia virus challenge. Journal of Virology. 82(7): 3517-3529.

  5. Bianchini, J., Simons, X., Humblet, M.F. and Saegerman, C. (2023). Lumpy skin disease: A systematic review of mode of transmission, risk of emergence and risk entry pathway. Viruses. 15(8): 1622. 

  6. Chervyakova, O.V., Zaitsev, V.L., Iskakov, B.K., Tailakova, E.T., Strochkov, V.M., Sultankulova, K.T., Sandybayev, N.T., Stanbekova, G.E., Beisenov, D.K., Abduraimov, Y.O. and Mambetaliyev, M. (2016) Recombinant sheep pox virus proteins elicit neutralizing antibodies. Viruses. 8(6): 159.

  7. Chung, C.S., Hsiao, J.C., Chang, Y.S. and Chang, W. (1998). A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. Journal of Virology. 72(2): 1577-1585. 

  8. Das, M., Chowdhury, M.S., Akter, S., Mondal, A.K., Uddin, M.J., Rahman, M.M., Rahman, M.M. (2021). An updated review on lumpy skin disease: Perspective of Southeast Asian countries. J. Adv. Biotechnol. Exp. Ther. Sep. 4(3): 322-33. 

  9. Dashprakash, M., Venkatesan, G., Ramakrishnan, M.A., Muthuchelvan, D., Sankar, M., Pandey, A.B., Mondal, B. (2015). Genetic diversity of fusion gene (ORF 117), an analogue of vaccinia virus A27L gene of capripox virus isolates. Virus Genes. 50(2): 325-328.

  10. Demkowicz, W.E., Maa, J.S. and Esteban, M. (1992). Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans. J. Virol. 66(1): 386-398.

  11. Gupta, T., Patial, V., Bali, D., Angaria, S., Sharma, M., Chahota ,R. (2020). A review: Lumpy skin disease and its emergence in India. Veterinary Research Communications. 44: 111-118.

  12. Hsiao, J.C., Chung, C.S. and Chang, W., (1998). Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: Identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain. Journal of Virology. 72(10): 8374-8379.

  13. King, A.M., Adams, M.J., Carstens, E.B. and Lefkowitz, E.J. (2012) Virus taxonomy. Classification and nomenclature of viruses. Ninth Report of the International Committee on Taxonomy of Viruses. 289-307.

  14. Kumar, A.R., Yogisharadhya, V., Bhanuprakash, G., Venkatesan. and Shivachandra, S.B. (2015). Structural analysis and immuno- genicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia like virus. Vaccine. 33(41): 5396-5405. 

  15. Manić, M., Stojiljković, M., Petrović, M., Nišavić, J., Bacić, D., Petrović, T. and Obrenovi, S. (2019) Epizootic features and control measures for lumpy skin disease in south east Serbia in 2016. Transboundary and Emerging Diseases. 66(5): 2087-2099. 

  16. Nan, W., Gong, M., Lu, Y., Li, J., Li, L., Qu, H., Liu, C., Wang, Y., Wu, F., Wu, X. and Wang, Z. (2023). A novel triplex real-time PCR assay for the differentiation of lumpy skin disease virus goatpox virus and sheeppox virus. Frontiers in Veterinary Science. 10: 1175391.

  17. Ntombela, N., Matsiela, M., Zuma, S., Hiralal, S., Naicker, L., Mokoena, N. and Khoza, T. (2023). Production of recombinant lumpy skin disease virus A27L and L1R proteins for application in diagnostics and vaccine development. Vaccine: X. 15: 100384.

  18. Ramírez, J.C., Tapia, E. and Esteban, M. (2002). Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions. Journal of General Virology. 83(5): 1059-1067.

  19. Rodriguez, J.F., Paez, E. and Esteban, M. (1987). A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers. Journal of Virology. 61(2):  395-404.

  20. Rudraraju, R. and Ramsay, A.J. (2010). Single-shot immunization with recombinant adenovirus encoding vaccinia virus glycoprotein A27L is protective against a virulent respiratory poxvirus infection. Vaccine. 28(31): 4997-5004. 

  21. Sanganagouda, K., Nagraja, K., Sajjanar, B., Kounin, S., Gomes, A.R., Pavithra, B.H., Lalasangi, S., Sumathi, B.R., Murag, S., Shankar, B.P. and KR, A.K. (2023). Molecular characterization, phylogenetic analysis and viral load quantification of Lumpy Skin Disease Virus in Cattle. 

  22. Singh, A.P., Dangi, N., Singh, P., Singh, V.K., Prabhu, S.N., Gangwar, N.K. and Biswas, S.K. (2024). Diagnostic utility of intranodular saline lavage in TaqMan probe-based real-time PCR diagnosis of lumpy skin disease. Emerging Animal Species. 10: 100037.

  23. Sodeik, B., Cudmore, S., Ericsson, M., Esteban, M., Niles, E.G. and Griffiths, G. (1995). Assembly of vaccinia virus: Incorporation of p14 and p32 into the membrane of the intracellular mature virus. Journal of Virology. 69(6): 3560-3574.

  24. Sprygin, A., Pestova, Y., Wallace, D.B., Tuppurainen, E. and Kononov, A.V. (2019). Transmission of lumpy skin disease virus: A short review. Virus Research. 269: 197637.

  25. Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Kutish, G.F. and Rock, D.L. (2001). Genome of lumpy skin disease virus. Journal of Virology. 75(15): 7122-7130. 

  26. Wang, H., Kong, Y., Mei, L., Lv, J., Wu, S., Lin, X. and Han, X. (2021). Multiplex real-time pcr method for simultaneous detection and differentiation of goat pox virus, sheeppox virus and lumpy skin disease virus. Journal of AOAC International. 104(5): 1389-1393.
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    Full Research Article

    Recombinant A27L Protein and Real-time PCR-based Detection of Lumpy Skin Disease Virus: A Step Toward Enhanced Diagnosis and Vaccine Research

    P
    Pravalika Annapureddy1
    V
    Vandana Gupta1,*
    G
    Gulshan Kumar1
    M
    Megha Katare Pandey2
    A
    Anju Nayak1
    S
    Swati Tripathi1
    R
    Renuka Mewade1
    S
    Sai Venkatesh Yakkali3
    1Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
    2Department of Translational Medicine, All India Institute of Medical Sciences Bhopal, Bhopal-462 026, Madhya Pradesh, India.
    3Department of Veterinary Pathology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.

    ABSTRACT

    Background: Lumpy skin disease (LSD) presents a significant threat to the global livestock industry, with potential implications for both animal health and economic stability. The recombinant A27L protein, derived from the lumpy skin disease Virus (LSDV), is a candidate for both diagnostic applications and vaccine development.

    Methods: In this study, we successfully cloned, expressed and purified the A27L protein using an E. coli expression system. A 447 bp fragment of the A27L gene was amplified from LSDV DNA, cloned into the pJET1.2/blunt vector and subcloned into the pQE30 expression vector. Expression of the recombinant protein was induced in E. coli M15 cells, yielding a 17.7 kDa protein, which was subsequently purified using nickel affinity chromatography. Additionally, a real-time PCR assay was performed for the differentiation of LSDV from other related poxviruses, enhancing the specificity of diagnostic testing.

    Result: Preliminary diagnostic validation of A27L protein was conducted using a DOT ELISA with LSDV-positive field serum sample, showing promising results. While the initial findings suggest the potential of the recombinant A27L protein for diagnostic use, further validation, including larger sample sizes and comparisons with existing diagnostic methods, is necessary to fully establish its effectiveness as a diagnostic tool and vaccine candidate. In addition, real-time PCR assay demonstrated specific detection and differentiation of LSDV from other related poxviruses.

    INTRODUCTION

    Lumpy skin disease (LSD), also known as Neethling virus disease, is a re-emerging, vector-borne disease of cattle, causing significant economic losses globally. Initially confined to sub-Saharan Africa, it has now become transboundary, with outbreaks reported in the Middle East, Europe, Russia and Asia (Babiuk et al., 2008). LSD is characterized by high fever (>104oF), multiple skin nodules, mucosal lesions, weight loss, lymphadenopathy, brisket edema and complications such as reduced milk yield, abortions, infertility, hide damage and secondary infections (Bedekovic et al., 2018). The disease is caused by the Neethling strain of Lumpy Skin Disease Virus (LSDV), a member of the genus Capripoxvirus, subfamily Chordo-poxvirinae, family Poxviridae (King et al., 2012). Transmission occurs via direct contact with infected secretions (nasal discharge, saliva, blood, milk, semen), intrauterine transmission and primarily through insect vectors like mosquitoes, ticks, flies and midges (Gupta et al., 2020; Sprygin et al., 2019). LSD typically shows high morbidity (up to 50%) and low mortality (<10%), though these rates vary with host immunity and vector prevalence (Das et al., 2021; Manić et al., 2019).
           
    LSDV is an enveloped virus with a dumbbell-shaped core and lateral bodies. Its 151 kbp double-stranded DNA genome includes 2.4 kbp inverted terminal repeats and encodes 156 ORFs, of which 146 are conserved and essential for replication, transcription and assembly (Tulman et al., 2001). Its 97% genomic similarity with Goatpox (GTV) and Sheeppox viruses (SPV) complicates differential serological diagnosis (Tulman et al., 2001; Bianchini et al., 2023). LSDV ORF 117 is an ortholog of the Vaccinia virus (VV) A27L protein, which is found on the surface of the intracellular mature virion (IMV) and plays a key role in virus-cell binding, membrane fusion and intracellular transport (Chung et al., 1998; Blouch et al., 2005). A27L is essential for viral morphogenesis, assembly and release, interacting with other viral proteins such as A17L to maintain structural integrity and infectivity (Kumar et al., 2015; Hsiao et al., 1998). The protein features a coiled-coil helical region that forms trimers, crucial for its functional activity (Rodriguez et al., 1987; Sodeik et al., 1995). Due to its role in immune evasion and infection, A27L has been identified as a potential antigen and target for neutralizing antibodies in poxviruses, including sheeppox and vaccinia virus (Chervyakova et al., 2016). It has demonstrated the ability to induce virus-neutralizing antibodies in mice and provides protective immunity when used as a vaccine target (Demkowicz et al., 1992; Berhanu et al., 2008; Rudraraju and Ramsay 2010). Monoclonal antibodies against A27L can neutralize viral infectivity, offering passive protection (Ramírez et al., 2002). Additionally, recombinant A27L proteins from various poxviruses are used for diagnostic purposes, including sero- monitoring and surveillance during outbreaks (Dashprakash et al., 2015; Chervyakova et al., 2016). In addition to protein-based diagnostics, Real-time PCR (qPCR) has become an invaluable tool for detecting and quantifying LSDV. Compared to conventional PCR, qPCR offers superior sensitivity, speed and reproducibility, making it an essential method for rapid diagnosis and timely intervention during outbreaks. By utilizing universal capripox primers and species-specific probes targeting conserved genetic regions, such as the RPO30 gene, qPCR allows for the differentiation of LSDV from other capripox viruses like Goatpox and Sheeppox (Wang et al., 2021). The use of qPCR in diagnostic protocols is vital for monitoring vaccine efficacy, controlling the spread of LSDV and conducting epidemiological studies, especially as capripox viruses continue to spread globally.
           
    This study aims to clone, express and purify the recom-binant A27L protein from LSDV and enhance diagnostic capabilities through differentiating real-time PCR. The combination of these two approaches holds promise for advancing the understanding and control of LSD, paving the way for better diagnostic tools and more effective vaccine development.

    MATERIALS AND METHODS

    Place of work and sample collection
     
    This study was carried out at the Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur, Madhya Pradesh from 2022-2024. Among the LSDV-positive tissue samples present in the department, one was selected for downstream cloning and expression of the target gene. In total, eleven LSDV tissue samples were screened using a real-time PCR assay for the detection of lumpy skin disease virus (LSDV). Subsequently, these samples were subjected to differential diagnosis to distinguish LSDV from goatpox virus.

    Genomic DNA extraction
     
    Genomic DNA from tissue samples and goat pox vaccine (Hester Biosciences Limited) was extracted using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s protocol.
     
    Gene amplification
     
    The extracted LSDV DNA was then subjected to PCR amplification using the GeneAmp PCR System 9700 (Applied Biosystems). The A27L gene fragment was amplified from the genomic DNA using the forward primer -AAT GGA TCC ATG GAC AGA GCT TTA TCA ATC TTT C and reverse primer R-AAT GTC GAC TCA TAG TGT TGT ACT TCG GCC, as described by (Ntombela et al., 2023) under the following PCR conditions: Initial denaturation at 95oC for 5 minutes, followed by 35 cycles consisting of denaturation at 95oC for 1 minute, annealing at 60°C for 40 seconds, extension at 72oC for 1 minute and a final elongation step at 72oC for 7 minutes. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel, using a 100 bp DNA ladder as a size marker.
     
    Cloning of A27 l gene
     
    The amplified PCR product for A27L gene was purified using Qiagen QIAquick® gel extraction kit (Qiagen, Germany) according to the manufacturer’s protocol. The cloning of the A27L gene using the pJET1.2/blunt vector was carried out as per the protocol provided by the CloneJET PCR Cloning Kit manufacturer. In order to insert the desired gene into the cloning vector, the purified PCR product with sticky ends needs to be converted into a blunt-ended form using DNA blunting enzyme. The blunt ended PCR product was ligated into the pJET1.2/blunt cloning vector using T4 DNA ligase enzyme. Following ligation, the recombinant plasmid, pJET1.2/blunt-A27L, was transformed into E. coli DH5α cells, which were selected on ampicillin-containing Luria Bertani (LB) agar plates. The resulting colonies were screened by colony PCR using both insert and vector primers (provided in the kit) to identify successful transformants and the PCR products were analyzed by agarose gel electrophoresis, confirming the presence of the 447 bp A27L fragment. To validate the cloning process, a positive control (provided in the kit) was utilized. Recombinant plasmid from the positive colonies was isolated using Favor PrepTM Plasmid Extraction Mini Kit (Favorgen, Taiwan). The recombinant plasmid (pJET1.2 and A27L) was digested with two restriction endonucleases, BamHI and Sal I. The digested PCR product was analysed by running it on a 1% agarose gel.
     
    Expression of A27L fusion protein
     
    The digested A27L gene and the pQE30 vector (BamHI and SalI restriction enzyme digested) fragments were purified using the Qiagen QIAquick® Gel Extraction Kit (Qiagen, Germany) according to the manufacturer’s protocol. Ligation of RE digested vector and insert was carried out using T4 DNA ligase. Recombinant plasmid vector pQE30-A27L was transformed into E. coli M15 cells and selected on ampicillin kanamycin containing LB agar plates. The colonies were screened for the presence of recombinant plasmid pQE30-A27L by colony PCR and also isolated recombinant plasmid was checked for insert release by double digestion with BamHI and Sal I restriction endonuclease followed by analysis of digested products on 0.8% agarose gel. For prokaryotic expression, recombinant bacterial clones containing the insert were cultured in 10 ml of LB broth supplemented with ampicillin (100 µg/ml) and kanamycin (50 µg/ml), with continuous shaking. Once the culture reached an optical density at 600 nm (OD 600) of 0.6 to 1.0, 1 mM IPTG was introduced to induce protein expression and the culture was incubated with constant shaking at room temperature. Two ml of the induced culture was collected every 2 hours starting from 2h onward up to 10 h. All the cultures collected were pelleted by centrifugation at 13,000 rpm and stored at -20oC for SDS-PAGE analysis.
           
    In this step, a positive control plasmid was also utilized. The control expression plasmid (pQE40), which encodes a 26 kDa protein, served as a positive control for expression, as outlined in the QIAexpress® Type IV Kit from Qiagen (Germany). This positive control is essential for validating the expression system and ensuring the reliability of the results obtained.
     
    Purification of expressed protein 
     
    The recombinant protein with histidine residues at N-terminal end of the protein was purified under denaturing conditions using Ni-NTA agarose. Around 250 ml culture was induced with 1 mM IPTG for 12 hrs under constant shaking at room temperature. The culture was pelleted by centrifugation at 13,000 rpm for 20 minutes. The obtained bacterial pellet was resuspended in 10 ml lysis buffer (8 M urea, 0.1 M NaH2PO4, pH 8.0). Incubation was carried out at room temperature for 1 hr. by gently swirling the cell suspension. Lysis was complete when the suspension gets translucent. Lysate was centrifuged at 14,000 rpm for 30 min at room temperature to pellet the cellular debris. Cell lysate supernatant containing the recombinant protein was applied to polypropylene column filled with one ml of Ni-NTA agarose. Flow through fraction was collected. Column was washed 2 times with 4 ml of wash buffer (8 M urea, 0.1 M NaH2PO4, pH 6.3). Wash fractions were also collected. 6x His-tagged protein was eluted three times with 0.5 ml of elution buffer D (8 M urea, 0.1 M NaH2PO4, pH 5.9) and collected as 500 µl aliquots.  Second elution was done three times with 0.5 ml of elution buffer E (8 M urea, 0.1 M NaH2PO4, pH 4.5) and collected as 500 µl aliquots. Twenty microlitre of each sample was mixed with equal volume of 2X SDS-PAGE sample buffer and stored at -20oC for SDS-PAGE analysis. The purified protein was estimated by lowry method.
     
    DOT ELISA for purified protein
     
    A 10 µL drop of peptide solution, with a concentration of 108 ng/µL, was applied onto a nitrocellulose membrane and allowed to dry for 10 minutes at room temperature. A negative control with PBS (Phosphate buffered saline) was used. Following the drying step, the membrane underwent three rounds of washing, each lasting 2 minutes, using a 0.1% solution of PBS-T (Phosphate-buffered saline with 0.05% Tween-20). Subsequently, the membrane was subjected to a blocking step by incubating it at 37oC for 1.5 hours with a blocking solution containing 5% bovine serum albumin (BSA) and 0.05% PBS-T. After the blocking procedure, the membrane was washed three times. For primary antibody incubation, LSDV-positive field serum was applied as the primary antibody and incubated for 45 minutes at 37oC. Following three additional washes with PBS-T, a secondary antibody conjugated to horseradish peroxidase (HRP), specifically anti-bovine HRP, was applied at a dilution of 1:2000. This incubation was carried out for 40 minutes at 37oC. After the final three washes, color development was initiated by preparing a reaction mixture consisting of 6 mg of 3, 3’-diaminobenzidine (DAB) dissolved in 10 mL of PBS, to which 10 µL of hydrogen peroxide was added. The reaction was stopped by the addition of an excess amount of double-distilled water.
     
    Real time PCR
     
    Real-time PCR (qPCR) was conducted on eleven LSDV-positive tissue samples from the Department of Veterinary Microbiology, NDVSU, Jabalpur. Amplification was performed using universal Capripoxvirus primers and an LSDV-specific probe on the QuantStudio™ 5 Real-Time Detection System (Applied Biosystems, Thermo Fisher Scientific). Data were analyzed using QuantStudio™ Design and Analysis Software (version 1.3.1). The Neoscript One-Step qRT-PCR Kit (Genes2Me, India) was used for all reactions, supplying the necessary reagents for efficient amplification and detection, as per the manufacturer’s protocol.
           
    The universal Capripoxvirus primers used for the real time amplification were -forward: 5′-ATG GTA GGA TAG TCG CAA ATG AT-3′  and reverse: 5′ -AGA TAT AAA CCC GGC AAG TGA C-3′ -along with species-specific probes: LSDV probe (5′ -FAM-TAA GCG ATT TTA TAG TTG CAA TGC GTA GT-BHQ-3′) and GTPV/SPPV probe (5′ -SUN-TAA GCG ATT TTA TAG TTG CGA TGC GTG GC-BkFQ-3′). Each 20 µl qPCR reaction contained 1.0 µl of each primer, 2.0 µl of template DNA, 1.0 µl of LSDV probe, 10.0 µl of master mix and 5.0 µl of nuclease-free water. Amplification was carried out in 0.2 ml PCR tubes using the following thermocycling conditions: initial denaturation at 95oC for 5 min, followed by 40 cycles of 95oC for 15 sec and 61oC for 35 sec (Wang et al., 2021).
           
    To differentiate LSDV from related Capripoxviruses (GTPV/SPPV), the assay was further optimized by testing each viral DNA template with both probes. No cross-reactivity was observed, confirming probe specificity. Duplex qPCR assays were then developed using both probes and both templates in a single 25 µl reaction (12.5 µl master mix, 0.5 µl enzyme, 2.0 µl each primer, 1.0 µl each probe, 1.5 µl each template and 3.0 µl nuclease-free water). This setup enabled simultaneous detection and differentiation of LSDV and GTPV/SPPV in a single assay, demonstrating high specificity and utility for field diagnostics.

    RESULTS AND DISCUSSION

    Amplification and cloning of LSDV A27L
     
    The A27L gene fragment from LSDV was successfully amplified, yielding a 447 bp amplicon (Fig 1). The PCR product was purified from an agarose gel and subsequently made blunt ended to facilitate ligation into the pJET1.2/blunt cloning vector. The ligation mixture was then transformed into E. coli DH5α competent cells (Fig 2). Transformants were screened by colony PCR using both insert-specific and vector-specific primers. PCR products amplified using the insert primers were analyzed by 1% agarose gel electrophoresis, confirming the presence of the expected 447 bp A27L fragment in positive colonies.

    Fig 1: Agarose gel electrophoresis showing amplified A27L Fusion gene.



    Fig 2: Transformation of plasmid (pJET1.2 + A27L) recombinant into E. coli DH5α cells.


           
    The PCR products using vector primers resulted a product size of 550 bp. In the process of amplifying a recombinant plasmid using vector-specific primers, the PCR amplifies not only the insert but also segments of the vector flanking the insertion site. Hence, the size of the amplicon was greater (550bp) than the size of the insert (447 bp) alone. Colony PCR was also performed for positive control (provided in the kit), resulting in the amplification of a DNA fragment with a size of 976 bp (Fig 3). This size is consistent with the specifications provided in the cloning kit manual (CloneJET PCR Cloning Kit), confirming the successful incorporation of the insert. After confirmation through colony PCR, recombinant plasmid (pJET1.2/blunt+ A27L) from the colonies was isolated and RE digested using BamHI and Sal I restriction endonucleases.

    Fig 3: Colony PCR using vector primers after transformation in pJET1.2 vector.


     
    Expression and purification of A27L fusion protein
     
    The restriction enzyme (RE)-digested A27L gene was ligated into the similarly digested pQE30 expression vector to form the recombinant construct pQE30-A27L. The plasmid was transformed into E. coli M15 cells and plated onto LB agar containing ampicillin and kanamycin for dual antibiotic selection (Fig 4). Transformants were screened by colony PCR using A27L-specific primers and the positive clones provided the expected 447 bp amplicon (Fig 5), confirming the successful insertion of the A27L gene into the expression vector. This two-vector cloning approach was deliberately employed to ensure greater precision and efficiency in expression. While direct cloning into the pQE30 expression vector is feasible, the use of pJET1.2/blunt as an intermediate vector provided an additional layer of monitoring.

    Fig 4: Growth of recombinant (pQE30 + insert) M15 cells in LB agar containing ampicillin and kanamycin.



    Fig 5: Colony PCR amplification of A27L fusion gene visualized by agarose gel electrophoresis.


          
    The positive recombinant colonies were induced with 1/ mM IPTG to initiate protein expression. While not fully induced, expression of the 17.7/ kDa A27L protein could be detected as early as 2 hours after induction, with band intensity progressively increasing up to 6 hours (Fig 6). The positive control (pQE40 plasmid) detected a clear band at approximately 26/ kDa, demonstrating that the positive control was functional expression system. Uninduced cells and control cultures did not exhibit visual additional bands, confirming with assurance that no leaky or non-specific expression was occurring. The cloning and expression of the A27L gene resulted in the production of a fusion protein with a size of 17.7 kDa. This finding is consistent with the work of (Ntombela et al., 2023), who reported an expressed protein of 21 kDa. The observed variation in protein size can be attributed to the different expression vectors utilized in the respective studies. In our research, we employed the pQE-30 vector, while (Ntombela et al., 2023), utilized the pET28a vector for cloning and expression. The differences in vector design and features can influence the processing and translation of the protein, leading to variations in the final protein size. The expressed recombinant A27L protein was purified from bacterial lysates with Ni-NTA metal chelate affinity chromatography. Following purification, a single sharp band at 17.7/ kDa that corresponded to A27L was clearly visualized on 12% SDS-PAGE, confirming confirmation that the target protein was successfully recovered and pure (Fig 7).

    Fig 6: SDS PAGE before purification.



    Fig 7: SDS PAGE after purification.


     
    DOT ELISA
     
    The immunogenicity of the purified recombinant protein was subsequently assessed using DOT ELISA. The expressed protein reacted with LSDV-positive field serum, producing a visible color change, while the negative control (PBS) showed no reaction (Fig 8). This demonstrates that the recombinant protein is specifically recognized by antibodies generated during LSDV infection, indicating that the A27L protein contains immunodominant epitopes. Immunoreactivity observed here aligns with the fundamental principle that recombinant viral proteins, when expressed correctly, can mimic native viral antigens and elicit specific antibody recognition. The absence of cross-reactivity with negative controls strengthens the reliability of this assay. These findings suggest that the recombinant A27L protein could serve as a candidate antigen for serological testing in diagnostic applications. However, broader validation with a larger panel of serum samples, including those from animals infected with other pathogens, will be essential to confirm specificity and rule out cross-reactivity.

    Fig 8: DOT ELISA for purified recombinant fusion protein.


     
    Real time PCR
     
    Real-time PCR analysis of eleven LSDV-positive tissue samples using universal Capripoxvirus primers and LSDV-specific probes yielded cycle threshold (Ct) values ranging from 13.905 to 20.538 (Fig 9). These values are in agreement with (Singh et al., 2024), who reported mean Ct values of 23.94±2.97 for skin biopsy samples and 25.23±2.41 for intranodular saline lavage and with (Sanganagouda et al., 2023), who observed Ct values between 6.81 and 23.23 in skin swabs. The relatively lower Ct values observed in this study suggest a higher viral load, reflecting the high sensitivity of real-time PCR for LSDV detection. These findings support the utility of real-time PCR in timely diagnosis and outbreak monitoring. Our results also align with (Wang et al., 2021), who noted that LSDV Ct values typically fall below 30. Such consistency across studies reinforces the robustness of the assay across different sample types and viral loads, underscoring its value in both clinical diagnostics and epidemiological surveillance.

    Fig 9: Amplification of Lumpy skin disease virus using real-time PCR, Ct values ranging from 13.905 to 20.538.


           
    A single-reaction real-time PCR assay was developed using both LSDV and GTPV templates, universal Capripox primers in combination with species-specific probes for LSDV and SPPV/GTPV. This assay successfully differentiated LSDV from GTPV, with the SPPV/GTPV probe amplifying GTPV at a Ct value of 18.096, while the LSDV probe detected LSDV at a significantly lower Ct value of 11.531 (Fig 10).

    Fig 10: Real time PCR using both probes and both templates, GTPV with a Ct value of 18.096 (Target 1), LSDV with a Ct value of 11.531 (Target 2).


           
    These findings are consistent with (Wang et al., 2021) and (Nan et al., 2023), who demonstrated that the GTPV/SPPV probe specifically detects GTPV DNA without amplifying LSDV and vice versa for the LSDV probe. The results validate the specificity and diagnostic effectiveness of the real-time PCR assay for differentiating Capripoxvirus infections.

    CONCLUSION

    This study successfully demonstrated the cloning, expression, and purification of the recombinant A27L protein from Lumpy Skin Disease virus, with preliminary findings showing its immunogenicity through specific recognition by LSDV-positive field serum samples, indicating potential diagnostic utility. However, the current results are preliminary due to limited sample size and lack of statistical validation. Further studies involving larger sample sets, control groups, cross-reactivity testing, and animal model evaluations are required to confirm its diagnostic specificity, sensitivity, and vaccine potential. Additionally, the GTPV/SPPV and LSDV probes showed high specificity by exclusively detecting their respective viral DNAs without cross-reactivity, supporting their usefulness in differentiating LSDV, GTPV, and SPPV infections for accurate diagnostics.

    CONFLICT OF INTEREST

    All authors declare that they have no conflicts of interest.

    REFERENCES


    1. Babiuk, S., Bowden, T.R., Boyle, D.B., Wallace, D.B. and Kitching, R.P. (2008). Capripoxviruses: An emerging worldwide threat to sheep, goats and cattle. Transboundary and Emerging Diseases. 55(7): 263-272.  

    2. Bedekovic, T., Simic, I., Kresic, N. and Lojkic, I. (2018). Detection of lumpy skin disease virus in skin lesions, blood, nasal swabs and milk following preventive vaccination. Transboundary and Emerging Diseases. 65(2): 491-496. 

    3. Blouch, R.E., Byrd, C.M. and Hruby, D.E. (2005). Importance of disulphide bonds for vaccinia virus L1R protein function. Virology Journal. 2: 1-5.

    4. Berhanu, A., Wilson, R.L., Kirkwood-Watts, D.L., King, D.S., Warren, T.K., Lund, S.A., Brown, L.L., Krupkin, A,K., VanderMay, E., Weimers, W. and Honeychurch, K.M. (2008). Vaccination of BALB/c mice with Escherichia coliexpressed vaccinia virus proteins A27L, B5R and D8L protects mice from lethal vaccinia virus challenge. Journal of Virology. 82(7): 3517-3529.

    5. Bianchini, J., Simons, X., Humblet, M.F. and Saegerman, C. (2023). Lumpy skin disease: A systematic review of mode of transmission, risk of emergence and risk entry pathway. Viruses. 15(8): 1622. 

    6. Chervyakova, O.V., Zaitsev, V.L., Iskakov, B.K., Tailakova, E.T., Strochkov, V.M., Sultankulova, K.T., Sandybayev, N.T., Stanbekova, G.E., Beisenov, D.K., Abduraimov, Y.O. and Mambetaliyev, M. (2016) Recombinant sheep pox virus proteins elicit neutralizing antibodies. Viruses. 8(6): 159.

    7. Chung, C.S., Hsiao, J.C., Chang, Y.S. and Chang, W. (1998). A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. Journal of Virology. 72(2): 1577-1585. 

    8. Das, M., Chowdhury, M.S., Akter, S., Mondal, A.K., Uddin, M.J., Rahman, M.M., Rahman, M.M. (2021). An updated review on lumpy skin disease: Perspective of Southeast Asian countries. J. Adv. Biotechnol. Exp. Ther. Sep. 4(3): 322-33. 

    9. Dashprakash, M., Venkatesan, G., Ramakrishnan, M.A., Muthuchelvan, D., Sankar, M., Pandey, A.B., Mondal, B. (2015). Genetic diversity of fusion gene (ORF 117), an analogue of vaccinia virus A27L gene of capripox virus isolates. Virus Genes. 50(2): 325-328.

    10. Demkowicz, W.E., Maa, J.S. and Esteban, M. (1992). Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans. J. Virol. 66(1): 386-398.

    11. Gupta, T., Patial, V., Bali, D., Angaria, S., Sharma, M., Chahota ,R. (2020). A review: Lumpy skin disease and its emergence in India. Veterinary Research Communications. 44: 111-118.

    12. Hsiao, J.C., Chung, C.S. and Chang, W., (1998). Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: Identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain. Journal of Virology. 72(10): 8374-8379.

    13. King, A.M., Adams, M.J., Carstens, E.B. and Lefkowitz, E.J. (2012) Virus taxonomy. Classification and nomenclature of viruses. Ninth Report of the International Committee on Taxonomy of Viruses. 289-307.

    14. Kumar, A.R., Yogisharadhya, V., Bhanuprakash, G., Venkatesan. and Shivachandra, S.B. (2015). Structural analysis and immuno- genicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia like virus. Vaccine. 33(41): 5396-5405. 

    15. Manić, M., Stojiljković, M., Petrović, M., Nišavić, J., Bacić, D., Petrović, T. and Obrenovi, S. (2019) Epizootic features and control measures for lumpy skin disease in south east Serbia in 2016. Transboundary and Emerging Diseases. 66(5): 2087-2099. 

    16. Nan, W., Gong, M., Lu, Y., Li, J., Li, L., Qu, H., Liu, C., Wang, Y., Wu, F., Wu, X. and Wang, Z. (2023). A novel triplex real-time PCR assay for the differentiation of lumpy skin disease virus goatpox virus and sheeppox virus. Frontiers in Veterinary Science. 10: 1175391.

    17. Ntombela, N., Matsiela, M., Zuma, S., Hiralal, S., Naicker, L., Mokoena, N. and Khoza, T. (2023). Production of recombinant lumpy skin disease virus A27L and L1R proteins for application in diagnostics and vaccine development. Vaccine: X. 15: 100384.

    18. Ramírez, J.C., Tapia, E. and Esteban, M. (2002). Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions. Journal of General Virology. 83(5): 1059-1067.

    19. Rodriguez, J.F., Paez, E. and Esteban, M. (1987). A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers. Journal of Virology. 61(2):  395-404.

    20. Rudraraju, R. and Ramsay, A.J. (2010). Single-shot immunization with recombinant adenovirus encoding vaccinia virus glycoprotein A27L is protective against a virulent respiratory poxvirus infection. Vaccine. 28(31): 4997-5004. 

    21. Sanganagouda, K., Nagraja, K., Sajjanar, B., Kounin, S., Gomes, A.R., Pavithra, B.H., Lalasangi, S., Sumathi, B.R., Murag, S., Shankar, B.P. and KR, A.K. (2023). Molecular characterization, phylogenetic analysis and viral load quantification of Lumpy Skin Disease Virus in Cattle. 

    22. Singh, A.P., Dangi, N., Singh, P., Singh, V.K., Prabhu, S.N., Gangwar, N.K. and Biswas, S.K. (2024). Diagnostic utility of intranodular saline lavage in TaqMan probe-based real-time PCR diagnosis of lumpy skin disease. Emerging Animal Species. 10: 100037.

    23. Sodeik, B., Cudmore, S., Ericsson, M., Esteban, M., Niles, E.G. and Griffiths, G. (1995). Assembly of vaccinia virus: Incorporation of p14 and p32 into the membrane of the intracellular mature virus. Journal of Virology. 69(6): 3560-3574.

    24. Sprygin, A., Pestova, Y., Wallace, D.B., Tuppurainen, E. and Kononov, A.V. (2019). Transmission of lumpy skin disease virus: A short review. Virus Research. 269: 197637.

    25. Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Kutish, G.F. and Rock, D.L. (2001). Genome of lumpy skin disease virus. Journal of Virology. 75(15): 7122-7130. 

    26. Wang, H., Kong, Y., Mei, L., Lv, J., Wu, S., Lin, X. and Han, X. (2021). Multiplex real-time pcr method for simultaneous detection and differentiation of goat pox virus, sheeppox virus and lumpy skin disease virus. Journal of AOAC International. 104(5): 1389-1393.
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