Molecular epidemiological analysis of lumpy skin disease virus isolates from recent outbreaks in India

Lumpy skin disease virus (LSDV) is an emerging transboundary pathogen causing severe economic losses in cattle. Continuous molecular surveillance is essential to understand the evolution and spread of circulating strains in India. Clinical samples, including nasal swabs, blood, and tissue specimens, were collected from suspected cattle during the 2025 outbreaks in Maharashtra and Karnataka. Samples were screened using the p32 gene PCR, virus isolation, followed by GPCR gene amplification, sequencing, and phylogenetic analysis. LSDV was detected in 33/40 of the samples. Nasal swabs showed the highest diagnostic sensitivity (95%), followed by tissue biopsies (82%). GPCR -based phylogenetic analysis revealed that the 2025 isolates formed a highly homogeneous clade closely related to the 2022 Haryana strain and distinct from earlier Indian and vaccine-associated lineages. Conserved amino acid substitutions (P34T and P61T) were identified in all isolates. The study demonstrates the circulation of a genetically stable yet evolving LSDV lineage in India and highlights the importance of targeted diagnostics and continuous genomic surveillance for effective outbreak monitoring and disease control.

Lumpy skin disease (LSD) is a highly contagious transboundary viral disease of cattle and buffaloes caused by the lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus within the family Poxviridae. The disease is characterized clinically by fever, enlarged superficial lymph nodes, nasal discharge, ocular discharge, edema, and the development of characteristic cutaneous nodules distributed over the body. These lesions often result in severe debilitation, reduced feed intake, mastitis, infertility, abortion, secondary bacterial infections, and permanent hide damage, leading to substantial economic losses to the livestock industry. In dairy animals, the disease significantly reduces milk production and animal productivity, thereby affecting the livelihood of smallholder farmers and the sustainability of the livestock sector (16, 1).

Historically, LSD was first reported in Zambia in 1929 and remained confined to sub-Saharan Africa for several decades before spreading rapidly across the Middle East, Europe, and Asia. Since 2013, the disease has shown unprecedented geographic expansion, facilitated by increasing transboundary animal movement, vector abundance, and changing ecological conditions. The disease is primarily transmitted mechanically through hematophagous arthropods, including mosquitoes, biting flies, and ticks, which play a critical role in the maintenance and dissemination of the virus under tropical and subtropical climatic conditions. In addition to vector-mediated transmission, direct or indirect contact through contaminated feed, water, equipment, and unrestricted animal movement contributes to disease spread (2).

The emergence of LSD in Asia has raised major concerns because the region harbors nearly 40% of the global cattle and buffalo population. India alone possesses one of the world’s largest livestock populations, accounting for approximately 536.76 million livestock animals, including nearly 300 million bovines (3). The livestock sector contributes significantly to the national economy, accounting for approximately 6.2% of the national gross domestic product (GDP) and nearly one-third of the agricultural GDP. Therefore, outbreaks of LSD have severe socio-economic implications due to mortality, decreased productivity, treatment costs, trade restrictions, and disruptions in livestock movement (4).

In India, LSD was first officially reported during August 2019 in the state of Odisha, particularly in the districts of Mayurbhanj, Bhadrak, and Kendrapara (5). Since then, the disease has spread rapidly across several states, causing extensive outbreaks with high morbidity and varying mortality rates. Subsequent epidemics affected millions of cattle across different agro-climatic regions of the country, highlighting the endemic establishment of the virus. Recent reports have also demonstrated spillover infections in atypical bovine hosts such as Mithun (Bos frontalis) and Yak (Bos grunniens), indicating possible expansion of host range and ecological adaptation of the virus (6, 7). The rapid dissemination and persistence of LSDV in India underscore the need for continuous epidemiological and molecular monitoring.

Africa and Asia were among the nations with the highest incidence of LSD globally in 2016–2020, and the epidemiology fluctuated significantly over time. The disease is primarily found in Asia, Africa, the Middle East, and some parts of Europe, but it has also been reported from numerous other regions of the world, including Iraq (8). Because of its high transmissibility and substantial economic impact, the World Organisation for Animal Health (WOAH) classifies LSD as a notifiable transboundary disease requiring immediate surveillance and control measures. The epidemiology of LSD has evolved considerably in recent years, with reports of emerging recombinant strains, vaccine-like variants, and genetically distinct field isolates from different geographic regions. Such evolutionary dynamics may influence viral virulence, host adaptation, transmission efficiency, and vaccine effectiveness. Consequently, genomic surveillance has become an essential component for understanding the molecular epidemiology and evolutionary trends of LSDV.

Among the available molecular markers, the G-protein-coupled chemokine receptor (GPCR) gene has been widely utilized for phylogenetic and evolutionary studies of LSDV due to its relatively high genetic variability and discriminatory capacity. Sequence analysis of the GPCR gene enables differentiation between classical field strains, recombinant strains, vaccine-associated strains, and geographically distinct lineages (9). Several studies have successfully employed GPCR-based phylogenetic analysis to investigate the origins of outbreaks, transmission dynamics, and lineage diversification of LSDV across endemic and newly affected regions.

Therefore, the present study was undertaken to molecularly characterize the LSDV strains circulating during the 2025 outbreaks in India using the GPCR gene, investigate their phylogenetic relationship with historical Indian and global LSDV strains, and evaluate the diagnostic efficiency of different clinical sample types for molecular detection of the virus. The findings of this study provide important insights into the ongoing evolution, transmission dynamics, and molecular epidemiology of LSDV circulating in India, contributing to the strengthening of future surveillance and control strategies.

Study area and sample collection

During the LSD outbreaks, field investigations were conducted in the affected regions of Maharashtra and Karnataka (Figure 1) with approval from the Department of Animal Husbandry and Veterinary Sciences (DAHD). Sampling was performed in accordance with guidelines prescribed by the WHO.

A total of 30 clinically suspected cattle showing nodular lesions, fever, and lymphadenopathy were included in the study. Multiple clinical specimens were collected from individual animals, resulting in a cumulative total of 40 samples comprising nasal swabs (n = 20), whole blood (n = 9), and tissue samples (n = 11), including skin scabs, lung, and liver. Nasal swabs were collected using sterile swabs and placed in viral transport medium (VTM). Blood samples (2–3 mL) were collected in EDTA vacutainers. Tissue samples were collected aseptically following proper disinfection procedures. All samples were transported under cold chain conditions and processed within 4–8 hrs in a BSL-2+ laboratory.

Ethics statement

The study was conducted in accordance with the guidelines of the World Organisation for Animal Health (WOAH). All procedures involving animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), ICAR-NIVEDI (Approval No. NIVEDI/IAEC/2022/06).

Sample processing

The collected samples were first thawed at room temperature before processing. Nasal swab samples were vigorously vortexed and aliquoted for subsequent DNA extraction. Blood samples (150 μL) were diluted with 50 μL of 1X phosphate-buffered saline (Himedia Cat: M1866 PBS; pH 7.2). Approximately 30 mg of tissue samples were aseptically minced using a sterile Bard-Parker blade. The tissue homogenates were then prepared by triturating the minced samples in 1 mL of chilled 1X PBS with the aid of a mortar and pestle.

Genomic DNA was extracted from all processed clinical specimens using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. The quality and concentration of the extracted DNA were determined using a Nanodrop spectrophotometer (Microdigital Co., Ltd., South Korea). Purified DNA samples were preserved at −20 °C until further analysis. Disposal of all biological waste was carried out following the standard operating procedures approved by the Institute Biosafety Committee (IBSC).

For molecular detection, the extracted genomic DNA was screened by PCR targeting the P32 gene (237 bp) to confirm Capripoxvirus infection (Table 1). PCR amplification of the P32 gene was performed in a total reaction volume of 25 µL containing 12.5 µL of 2X Green Taq PCR Master Mix (Thermo Scientific, USA), 0.5 µL each of forward and reverse primers (10 pmol/µL), 3 µL of template DNA (~100 ng), and 8.5 µL of nuclease-free water. Amplification was carried out in a thermal cycler using the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 52 °C for 30 sec, and extension at 72 °C for 45 sec, with a final extension at 72 °C for 10 min. The reaction was held at 4 °C until further processing. PCR products were analyzed by electrophoresis on 1.5% agarose gel stained with ethidium bromide/alternative nucleic acid stain visualized under a UV transilluminator using a Gel Documentation System (Vilber Bio-Print,Vilber Lourmat, France).

Samples that tested positive for the P32 gene were subsequently subjected to virus isolation, except for blood.

PCR amplification of the GPCR gene (1200 bp) was carried out in a total reaction volume of 25 µL containing 12.5 µL of 2X Green Taq PCR Master Mix (Thermo Scientific, USA), 0.5 µL each of forward and reverse primers (10 pmol/µL), 3 µL of template DNA (~100 ng), and 8.5 µL of nuclease-free water. Amplification was performed in a thermal cycler under the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min 30 sec, with a final extension at 72 °C for 10 min. The reaction was held at 4 °C until further processing.

Virus isolation

The Madin-Darby Bovine Kidney (MDBK) cell line was used for virus adaptation and isolation. Nasal swab samples and tissue suspensions that tested positive by PCR were syringe- filtered using a 0.45 μm filter and subsequently used to infect a confluent monolayer of MDBK cells. Virus-infected MDBK cell lines were maintained in Minimum Essential Medium (MEM; HiMedia, Mumbai, India) supplemented with an antibiotic-antimycotic solution (1X; HiMedia, Mumbai, India) and 2% fetal bovine serum (FBS; MP Biomedicals, Santa Ana, CA, USA). The infected cells were incubated at 37 °C in a 5% CO 2 incubator (Thermo Scientific, Waltham, MA, USA) and monitored daily for five days to observe cytopathic effects (CPE). Following infection, the virus was harvested by subjecting it to three freeze-thaw cycles and stored at -80 °C. The viral suspension was collected and passaged until the appearance of CPE. DNA extracted from the harvested virus was further confirmed by PCR targeting the GPCR gene.

Sequencing

PCR-positive amplicons of the GPCR (G-protein coupled receptor) gene were resolved by agarose gel electrophoresis, excised, and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The purified DNA was stored at −20 °C and subsequently subjected to Sanger sequencing at Eurofins Genomics India Private Limited, Bengaluru, India.

Sequencing data were analyzed and edited using GeneTool software (Informer Technologies, Inc.), and the obtained sequences were submitted to the GenBank database and assigned accession numbers.

Phylogenetic analysis

Comparative sequence analysis was performed using Capripoxvirus-associated nucleotide sequences of the GPCR gene retrieved from GenBank, including in-house Karnataka isolates, sequences from other Indian states, representative global LSDV strains, and vaccine strains. Representative goatpox virus (GTPV) and sheeppox virus (SPPV) sequences were included as outgroup taxa to root the phylogenetic tree.

Multiple sequence alignment was carried out using the MUSCLE algorithm implemented in Molecular Evolutionary Genetics Analysis (MEGA) version 11. Phylogenetic relationships were inferred using the Maximum Likelihood (ML) method based on the Tamura 3-parameter nucleotide substitution model, selected as the most suitable model for the aligned dataset. Statistical support for branching patterns was evaluated using 1000 bootstrap replicates, and bootstrap values greater than 70% were indicated at the corresponding nodes. Initial trees for heuristic searching were generated automatically using the Neighbor-Joining (NJ) and BioNJ algorithms based on pairwise distance matrices. The topology with the highest log-likelihood score was selected as the final phylogenetic tree. The final tree was visualized using MEGA Tree Explorer and further refined, annotated, and graphically formatted using Interactive Tree of Life (iTOL) version 7.

Epidemiology of LSDV

According to the 20th Livestock Census (2019), Karnataka possesses a substantial bovine population, including approximately 8.46 million cattle, while Maharashtra also maintains a large livestock population, rendering both states highly susceptible to LSD outbreaks. In Karnataka, districts such as Belagavi, Hassan, and Shivamogga exhibited high cattle density, whereas major outbreaks in Maharashtra were predominantly reported from Jalgaon, Chandrapur, Pune, and Solapur districts.

Vaccination coverage against LSD in Karnataka increased markedly from 0.99% in 2021 to nearly complete coverage during 2022–2023, followed by a slight decline to 94.8% in 2024. Similarly, Maharashtra achieved approximately 93% vaccination coverage, with several districts reporting coverage exceeding 90%–95%. Despite extensive vaccination efforts, widespread disease transmission was observed in both states. Karnataka reported infection in more than two million cattle distributed across 4,380 villages and 160 taluks. In Maharashtra, approximately 98,000 cases were documented, with major clustering observed in Chandrapur district (approximately 50,000 cases) and the Nashik division (approximately 2,700 cases).

Temporal variation in case fatality rates (CFR) was also evident. In Karnataka, the CFR increased from 0.15% during 2021–2022 to 13.8% during 2023–2024, particularly in northern agro-climatic zones. In contrast, Maharashtra exhibited a comparatively lower overall CFR (0.3%–0.4%), although district-level fatality rates ranged between 0.5% and 3%. The observed epidemiological patterns indicate that factors such as cattle density, animal movement, vector abundance, and gaps in vaccination coverage may have contributed significantly to the persistence and spread of LSD in both states.

Virus isolation

Representative samples, including tissue suspensions and nasal swabs collected during LSD outbreaks, were subjected to virus isolation in MDBK cell lines. The isolates were serially passaged through two to three blind passages to facilitate viral adaptation and the development of CPE. Infected cells exhibited distinct morphological changes suggestive of LSDV infection, including cellular rounding, vacuolation, ballooning degeneration, multinucleated syncytia formation, and eventual cell detachment leading to cytolysis. The formation of localized foci containing clusters of infected cells further confirmed active viral replication. The field isolates were successfully adapted to the MDBK cell line, with prominent CPE becoming evident between the sixth and tenth passages. These observations demonstrate the strong cytopathic nature of LSDV and confirm successful virus isolation under in vitro conditions.

Molecular detection

During the peak of the outbreak, molecular screening of 40 clinical samples using P32 gene-based PCR confirmed Lumpy Skin Disease Virus (LSDV). Clinically affected cattle exhibited classical signs of LSD, including fever, anorexia, reduced milk yield, lymphadenopathy, lacrimation, hypersalivation, and characteristic nodular skin lesions (Figure 2).

PCR positivity varied among different sample types, with nasal swabs exhibiting the highest detection rate (95%,19/20), followed by tissue biopsies (82%, 9/11), whereas blood samples showed comparatively lower detection efficiency (55%, 5/9). The higher positivity observed in nasal swabs and lesion-derived samples suggests that LSDV is predominantly localized in respiratory secretions and cutaneous lesions during active infection rather than circulating extensively in blood at the time of sampling (Figure 3). These findings demonstrate that sample type significantly influences diagnostic sensitivity and indicate that nasal swabs and lesion-derived samples are more suitable for PCR-based detection of LSDV under field conditions, while blood samples may have comparatively limited diagnostic utility.

Phylogenetic analysis

The obtained GPCR gene sequences were analyzed and edited using GeneTool software (Informer Technologies, Inc.). Six representative sequences were submitted to the GenBank database and assigned accession numbers from PX898063 to PX898068.

Phylogenetic analysis of the GPCR gene demonstrated that the 2025 outbreak isolates clustered into a highly homogeneous and well-supported monophyletic clade (bootstrap value: 1000), indicating minimal genetic divergence among circulating strains (Figure 4). This tight clustering suggests a common and recent ancestral origin, consistent with rapid local transmission during the outbreak.

The 2025 isolates showed the highest nucleotide identity with the 2022 Haryana strain (OQ920898.1), confirming their placement within the contemporary Indian field strain lineage. The short branch lengths observed between these sequences further support limited accumulation of mutations over time, indicating relative genetic stability of the virus in recent years. In contrast, the study isolates were clearly segregated from earlier Indian strains, including those from the 2019 Jharkhand and 2020 Karnataka outbreaks. This separation reflects a temporal evolutionary shift, possibly driven by gradual mutation accumulation and regional adaptation, leading to the emergence of a more recent dominant lineage in India (Figure 4). Importantly, the 2025 isolates remained phylogenetically distant from recombinant strains and vaccine-derived lineages reported in other regions. This distinction confirms that the current outbreak is unlikely to be associated with vaccine spill over or recombination events, reinforcing its classification as a circulating wild-type field strain.

Overall, the phylogenetic pattern indicates ongoing circulation of a genetically stable and epidemiologically dominant LSDV lineage in India since at least 2022, with limited diversification but clear divergence from earlier outbreak strains. This highlights the importance of continuous genomic surveillance to monitor potential emergence of new variants or recombinant forms that could impact disease dynamics or vaccine efficacy.

Amino acid sequence analysis

Sequence analysis of the GPCR gene revealed conserved amino acid substitutions at positions P34T and P61T across all isolates, indicating potential lineage-specific signatures.

All sequences exhibited a high degree of nucleotide identity (>99%) among themselves, confirming the genetic homogeneity of the circulating isolates. Comparative analysis with reference strains showed that these substitutions were consistently present in the 2025 isolates but absent in earlier Indian strains, suggesting recent evolutionary divergence.

No insertions, deletions, or frameshift mutations were observed in the analyzed region, indicating structural stability of the GPCR gene. The identified substitutions were non-synonymous, leading to amino acid changes that may influence protein conformation or function.

Multiple sequence alignment demonstrated that the mutations were uniformly distributed across all samples, further supporting their role as stable molecular markers. Additionally, conserved regions flanking these mutations remained unchanged, highlighting the overall genetic conservation of the gene despite minor variations.

These findings suggest that the circulating isolates represent a genetically stable yet distinct lineage, with specific amino acid substitutions that can serve as reliable markers for molecular epidemiology and strain differentiation.

The present study provides comprehensive insights into the molecular epidemiology, diagnostic dynamics, and evolutionary patterns of Lumpy Skin Disease Virus (LSDV) during the 2025 outbreaks in India. The identification of a highly homogeneous cluster among the study isolates suggests the predominance of a single circulating strain, indicative of localized transmission and epidemiological stability. Such clustering is often associated with rapid outbreak expansion driven by a well-adapted viral lineage, rather than multiple independent introductions (12).

The close phylogenetic relationship of the 2025 isolates with the 2022 Haryana strain (OQ920898.1) strongly supports a north-to-west transmission trajectory. This pattern is likely influenced by uncontrolled livestock movement, informal trade networks, and the widespread distribution of competent arthropod vectors. Given the transboundary nature of LSD, such intra-country dissemination highlights gaps in movement control and emphasizes the need for strengthened surveillance at both regional and national levels.

The observed genetic divergence from earlier Indian isolates (Jharkhand, 2019 and Karnataka, 2020) and clear separation from vaccine-derived and recombinant strains indicate ongoing viral evolution under field conditions. This divergence may reflect adaptation to local ecological niches, host immune pressures, or vector dynamics. Importantly, the absence of clustering with vaccine-like strains suggests that the currently circulating viruses are field strains rather than vaccine spillover, which has been a concern in some regions globally (13).

The conserved amino acid substitutions (P34T and P61T) identified in the GPCR gene across all isolates may serve as molecular signatures of the emerging lineage. While their exact functional implications remain unclear, such substitutions could influence viral fitness, host interaction, or immune evasion. Similar lineage-specific mutations have been reported in capripoxviruses and are often associated with regional adaptation. However, functional validation through in vitro and in vivo studies is essential to establish their biological significance.

From a diagnostic standpoint, the study clearly demonstrates that sample selection critically influences PCR detection efficiency. The significantly higher positivity rates in nasal swabs and lesion-derived samples (scabs and tissue biopsies) confirm that LSDV exhibits strong tropism for epithelial and mucocutaneous tissues. The lower detection rate in blood samples is consistent with the transient nature of viremia in LSDV infections, which typically occurs during early stages and declines as the virus localizes in skin lesions. These findings are particularly relevant for field diagnostics, as they support the adoption of non-invasive and easily collectable samples such as nasal swabs for large-scale surveillance programs (14, 15).

The integration of molecular findings with epidemiological observations further strengthens the conclusions of this study. The dominance of a single lineage, coupled with its geographical spread, suggests that vector-mediated transmission and animal movement are key drivers of outbreak dynamics. Environmental factors such as temperature, humidity, and rainfall likely contribute to vector proliferation, thereby facilitating sustained transmission cycles. This aligns with previous studies that have identified seasonal and ecological determinants as critical factors influencing LSD outbreaks.

Another important implication of this study is the potential impact of viral evolution on disease severity and control strategies. Although vaccination campaigns have contributed to reducing overall mortality, the persistence and spread of a distinct lineage raise questions regarding vaccine efficacy against emerging variants. Continuous genomic monitoring is therefore essential to detect antigenic changes that may compromise vaccine-induced immunity. Additionally, the rising case fatality trends observed in some regions may be partially attributed to viral adaptation, host susceptibility, or delays in intervention.

Despite the valuable insights provided, certain limitations must be acknowledged. The relatively small sample size and geographic focus may not fully capture the diversity of circulating strains across India. Furthermore, reliance on partial gene sequencing (GPCR gene) limits the resolution of evolutionary analysis compared to whole-genome sequencing. Future studies incorporating larger datasets and full-genome analyses would provide a more comprehensive understanding of viral evolution and transmission dynamics.

Moving forward, there is a critical need to adopt an integrated “One Health” approach combining molecular surveillance, vector ecology, and livestock management practices. Advanced analytical tools, including phylogeographic modeling and machine learning-based prediction systems, could significantly enhance outbreak forecasting and response strategies. Additionally, studies focusing on host immune responses, breed susceptibility, and vaccine performance under field conditions will be essential for refining control measures.

In conclusion, this study highlights the emergence and dominance of a genetically distinct LSDV lineage in India during the 2025 outbreaks. The findings underscore the importance of targeted diagnostics, continuous genomic surveillance, and region-specific intervention strategies to effectively manage and control the spread of LSD.