Lumpy skin disease virus ORF127 protein suppresses type I interferon responses by inhibiting K63-linked ubiquitination of tank binding kinase 1.

Lumpy skin disease (LSD) is a severe animal infectious disease caused by lumpy skin disease virus (LSDV), inducing extensive nodules on the cattle mucosa or the scarfskin. LSDV genome encodes multiple proteins to evade host innate immune response. However, the underlying molecular mechanisms are poorly understood. In this study, we found that LSDV could suppress the expression of IFN-ß and interferon-stimulated genes (ISGs) in MDBK cells during the early stage of infection. Subsequently, an unbiased screen was performed to screen the LSDV genes with inhibitory effects on the type I interferon (IFN-I) production. ORF127 protein was identified as one of the strongest inhibitory effectors on the expression of IFN-ß and ISGs, meanwhile, the 1-43 aa of N-terminal of ORF127 played a vital role in suppressing the expression of IFN-ß. Overexpression of ORF127 could significantly promote LSDV replication through inhibiting the production of IFN-ß and ISGs in MDBK cells. Mechanism study showed that ORF127 specifically interacted with TBK1 and decreased the K63-linked polyubiquitination of TBK1 which suppressed the phosphorylation of TBK1 and ultimately decreased the production of IFN-ß. In addition, truncation mutation analysis indicated that the 1-43 aa of N-terminal of ORF127 protein was the key structural domain for its interaction with TBK1. In short, these results validated that ORF127 played a negative role in regulating IFN-ß expression through cGAS-STING signaling pathway. Taken together, this study clarified the molecular mechanism of ORF127 gene antagonizing IFN-I-mediated antiviral, which will helpfully provide new strategies for the treatment and prevention of LSD.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Label-Free and DNAzyme-Mediated Biosensor with a High Signal-to-Noise Ratio for a Lumpy Skin Disease Virus Assay.

Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·µL-1. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.

Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics.

IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Detection of lumpy skin disease virus reads in the human upper respiratory tract microbiome requires further investigation.

Lumpy skin disease virus (LSDV), a double-stranded DNA virus from the Capripoxvirus genus, primarily affects Bos indicus, Bos taurus breeds, and water buffalo. Arthropod vectors, including mosquitoes and biting flies, are the main LSDV transmitters. Although LSDV is not zoonotic, this study unexpectedly detected LSDV reads in the upper respiratory tract microbiome of humans from rural and urban areas in Maharashtra, India. Nasopharyngeal and oropharyngeal swab samples collected for SARS-CoV-2 surveillance underwent whole-genome metagenomics sequencing, revealing LSDV reads in 25% of samples. Split kmer analysis provided insights into sample relatedness despite the low coverage of LSDV reads with the reference genome. Our findings, which include the detection of LSDV contigs aligning to specific locations on the reference genome, suggest a common source for LSDV reads, potentially shared water sources, or milk/milk products. Further investigation is needed to ascertain the mode of transmission and reason for the detection of LSDV reads in human upper respiratory tract.

Lumpy skin disease: Insights into current status and geographical expansion of a transboundary viral disease.

Lumpy skin disease (LSD) is an emerging transboundary viral disease of livestock animals which was first reported in 1929 in Zambia. Although LSD is a neglected disease of economic importance, it extends a direct impact on the international trade and economy in livestock-dependent countries. Lumpy skin disease virus (LSDV) has been endemic in African countries, where several outbreaks have been reported previously. However, the virus has spread rapidly across the Middle East in the past two decades, reaching Russia and, recently, the Asian subcontinent. With unprecedented cluster outbreaks being reported across Asian countries like India, China, Nepal, Bangladesh, and Pakistan, LSDV is certainly undergoing an epidemiological shift and expanding its geographical footprint worldwide. Due to high mortality among livestock animals, the recent LSD outbreaks have gained attention from global regulatory authorities and raised serious concerns among epidemiologists and veterinary researchers. Despite networked global surveillance of the disease, recurrent LSD cases pose a threat to the livestock industry. Hence, this review provides recent insights into the LSDV biology by augmenting the latest literature associated with its pathogenesis, transmission, current intervention strategies, and economic implications. The review critically examines the changing epidemiological footprint of LSDV globally, especially in relation to developing countries of the Asian subcontinent. We also speculate the possible reasons contributing to the ongoing LSD outbreaks, including illegal animal trade, climate change, genetic recombination events between wild-type and vaccine strains, reversion of vaccine strains to virulent phenotype, and deficiencies in active monitoring during the COVID-19 pandemic.

Emerging evidence for poxvirus-mediated unfolded protein response: Lumpy skin disease virus maintains self-replication by activating PERK and IRE1 signaling.

The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK-eIF2α and IRE1-XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.

Detection and molecular characterization of lumpy skin disease and bovine papular stomatitis viruses in lumpy skin disease-suspected outbreaks in Tanzania.

BACKGROUND: Lumpy Skin Disease (LSD) is endemic in sub-Saharan countries and is currently a global threat to the cattle industry. Information on the circulating Capripoxvirus lumpyskinpox, formerly known as Lumpy Skin Disease Virus (LSDV), and other poxviruses infecting cattle is very scant in Tanzania. The current study aimed to confirm and characterize LSDV and other poxviruses infecting cattle, from LSD suspected outbreaks in Tanzania. METHODS: A total of 24 samples were collected from four LSD suspected outbreaks reported in Tanzania between February and May 2023. Samples were screened for LSDV genome by real-time PCR and then subjected to a high-resolution multiplex melting (HRM) assay where 10 samples were positive for Capripoxvirus (CaPV) and one sample was Parapoxvirus (PPV) positive. Four LSDV genes; RPO30, GPCR, EEV glycoprotein and B22R and the partial B2L gene of PPVs were analyzed. RESULTS: All targeted LSDV genes from the Tanzanian isolates showed 100% similarity and isolates clustered with commonly circulating LSDV field isolates. Furthermore, the single nucleotide polymorphism (SNP) at position 240 (A-> G) of the EEV gene differentiates the Tanzanian LSDVs from the group of ancient Kenyan LSDV isolates while the B22R sequences of the Tanzanian LSDV isolates differed from the LSDV Neethling and LSDV KSGP-0240 derived vaccines. Sequence analysis of the partial B2L gene of the Tanzanian parapoxvirus bovinestomatitis, formerly known as Bovine papular stomatitis virus (BPSV) showed a different BPSV strain circulating compared to publicly available sequences. CONCLUSION: These findings confirm the presence of LSDV in Tanzania, which suggesting the need for establishing an effective control program and continuous monitoring. The presence of a typical profile for Tanzania BPSV is an indication that, although never reported before, BPSV is established in the country therefore this virus should be included in the differential diagnosis of LSDV.

An insight into emergence of lumpy skin disease virus: a threat to Indian cattle.

Lumpy skin disease (LSD) is a highly infectious and economically devastating viral disease of cattle. It is caused by Lumpy Skin Disease Virus (LSDV) belonging to the genus Capripoxvirus and family Poxviridae. The origin of lumpy skin disease has been traced to Zambia, (an African nation) in Southern part during the year 1929. The first reported case of LSD besides Africa was from Israel, a Middle Eastern nation, thus proving inter-continental spread. Subsequently, the disease entered Middle East, Eastern Europe and Asia with numerous outbreaks in the recent years. LSD has emerged as a significant concern in the Indian sub-continent, due to outbreaks reported in countries such as Bangladesh, India, China in 2019. In the following years, other South and East Asian countries like Taipei, Nepal, Sri Lanka, Myanmar, Bhutan, Vietnam, Hong Kong, Thailand, Malaysia, Laos, Cambodia, Pakistan, Indonesia and Singapore also faced severe outbreaks. At present, LSD is considered to be an emerging disease in the Indian sub-continent due to the recent status of disease. Considering the global scenario, LSDV is changing its transmission dynamics as evidenced by a shift in its epidemiology. As a result of high morbidity and mortality rate among cattle, the current outbreaks have been a major cause of socio-economic catastrophe. This contagious viral disease has eminent repercussions as the estimated monetary damage incurred is quite high. Despite having networked surveillance and comprehensive databases, the recurring outbreaks have raised major concern among researchers. Therefore, this review offers brief insights into the emergence of LSDV by amalgamating the newest literature related to its biology, transmission, clinico-pathology, epidemiology, prevention strategies, and economic consequences. Additionally, we have also provided the epidemiological insights of the recent outbreaks with detailed state wise studies.

Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs.

Lumpy skin disease virus (LSDV) infection is a major socio-economic issue that seriously threatens the global cattle-farming industry. Here, a recombinant virus LSDV-ΔTK/EGFP, expressing enhanced green fluorescent protein (EGFP), was constructed with a homologous recombination system and applied to the high-throughput screening of antiviral drugs. LSDV-ΔTK/EGFP replicates in various kidney cell lines, consistent with wild-type LSDV. The cytopathic effect, viral particle morphology, and growth performance of LSDV-ΔTK/EGFP are consistent with those of wild-type LSDV. High-throughput screening allowed to identify several molecules that inhibit LSDV-ΔTK/EGFP replication. The strong inhibitory effect of theaflavin on LSDV was identified when 100 antiviral drugs were screened in vitro. An infection time analysis showed that theaflavin plays a role in the entry of LSDV into cells and in subsequent viral replication stages. The development of this recombinant virus will contribute to the development of LSDV-directed antiviral drugs and the study of viral replication and mechanisms of action.

Genome sequence characterization of the unique recombinant vaccine-like lumpy skin disease virus strain Kurgan/2018.

In 2018, the molecular epidemiology of lumpy skin disease in Russia was characterized by a surge in novel recombinant vaccine-like strains causing outbreaks along the southern border, spreading in an easterly direction. Currently, five distinct novel recombinant vaccine-like lineages have been described, designated as clusters 2.1 to 2.5. Based on the complete genome sequence analysis of the causative lumpy skin disease virus (Kurgan/Russia/2018), obtained from an eponymous outbreak, the genome was shown to be composed of a Neethling vaccine strain virus as the dominant parental strain and KSGPO vaccine virus as its minor parental strain. These features are similar to those of Saratov/Russia/2017 and Tyumen/Russia/2018, representing clusters 2.1 and 2.4, respectively. However, Kurgan/Russia/2018 has 16 statistically significant recombination events unique to this sequence, contributing to the phylogenetic clustering of Kurgan/Russia/2018 in yet another cluster designed cluster 2.6, based on analysis involving the complete genome sequences.

Emergence of lumpy skin disease virus (LSDV) infection in domestic Himalayan yaks (Bos grunniens) in Himachal Pradesh, India.

In this study, we investigated and confirmed natural lumpy skin disease virus (LSDV) infection in Himalayan yaks (Bos grunniens) in Himachal Pradesh, India, based on clinical manifestations and results of genome detection, antibody detection, virus isolation, and nucleotide sequencing. Subsequent phylogenetic analysis based on complete GPCR, RPO30, and EEV gene sequences revealed that the LSDV isolates from these yaks and local cattle belonged to LSDV subcluster 1.2.1 rather than the dominant subcluster 1.2.2, which is currently circulating in India, suggesting a separate recent introduction. This is the first report of natural LSDV infection in yaks in India, expanding the known host range of LSDV. Further investigations are needed to assess the impact of LSDV infection in yaks.

Isolation and molecular characterization of lumpy skin disease virus from Tamil Nadu, India during the outbreaks from 2020 to 2022.

Lumpy skin disease (LSD) caused by LSD virus is a WOAH notifiable, high-impact, transboundary poxviral disease of bovines. The first official report of LSDV in India is from Odisha state during August 2019. Since then, cases have been reported from many states including Tamil Nadu, a Southern state of India. The present study deals with isolation and molecular characterization of LSDV from Tamil Nadu during the period August 2020 to July 2022. LSDV was isolated in embryonated chicken eggs (ECE) and BHK 21 cells and was characterized based on P32, RPO30, and GPCR genes. The phylogenetic analysis revealed that Tamil Nadu isolates from India are closely related to other Indian strains, Kenyan strains and strains from neighboring countries such as Bangladesh, Nepal, and Myanmar confirming the common exotic source for the transboundary spread across borders. The presence of unique signature of amino acid (aa) at specific positions (A11, T12, T34, S99, and P199) in the GPCR sequence confirmed the identity of LSDV. A twelve nucleotide (nt94-105) insertion and corresponding aa (TILS) at 30-33 position was found in GPCR sequence and characteristic amino acid proline at 98 position (P98) in the RPO30 gene sequence of our isolates was similar to strains from Bangladesh, Nepal, and Myanmar. Further, dissimilarity of our isolates from Neethling like vaccine strains confirms the circulation of virulent filed strains responsible for the outbreaks.

Prediction of lumpy skin disease virus using customized CBAM-DenseNet-attention model.

Lumpy skin disease virus (LSDV) is an extremely infectious, viral, and chronic skin disease that is caused by the Capripox virus. This viral disease is predominantly found in cows. Mosquitoes and ticks are the primary transmitters for the spread of this virus. Recently, LSDV has been rapidly spreading all over the world, especially in several areas of Pakistan, India, and Iran. Thousands of cows have died due to this infectious virus in Pakistan and early detection of LSDV is needed to avoid further loss. The prediction and classification of LSDV are hindered by the lack of publicly available datasets. Despite a few studies using LSDV datasets, such datasets are often small, which may lead to model overfitting. In this regard, we collect the dataset from several online sources, as well as, collecting images from veterinary farms in different areas of Pakistan. Deep learning has been widely used in the medical field for disease detection and classification. Therefore, this study leverages DenseNet deep learning models for LSDV detection and classification. Experiments are performed using VGG-16, ResNet-50, MobileNet-V2, custom-designed convolutional neural network, and Inception-V3. The DenseNet architecture presents a Convolutional Block Attention Module (CBAM) and Spatial Attention (SA) for the prediction and classification of LSD. Results demonstrate that a 99.11% accuracy can be obtained on the augmented dataset while a 94.23% accuracy can be achieved with the original dataset for chicken pox, monkey pox, and LSDV. Comparison with state-of-the-art studies corroborates the superior performance of the proposed model.

Development of Paper-Based Fluorescent Molecularly Imprinted Polymer Sensor for Rapid Detection of Lumpy Skin Disease Virus.

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.

The expanding host range of lumpy skin disease virus in wild and domestic animals.

Clinical lumpy skin disease (LSD) predominantly affects cattle and, to lesser extent domestic water buffalos. Whilst earlier work focussed on the disease in Africa, the recent emergence of LSD virus (LSDV) as a major cause of disease in Asia has led to a widening range of susceptible hosts for the virus. This article lists the wild and domestic ungulates in which LSDV infection has been confirmed and considers the significance of the disease for these species in Asia.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Epidemiology, genetic characterization and population structure analysis of lumpy skin disease virus from North India.

Lumpy skin disease (LSD) is a transboundary, viral disease of cattle with a significant economic impact on the livestock industry. This study describes the epidemiological investigations of outbreaks of LSD that occurred in 2022 in three Indian states viz., Haryana, Himachal Pradesh and Rajasthan and the genetic characterization of Lumpy skin disease viruses (LSDVs). Also, the population structure analysis of LSDVs was carried out. Out of 138 scab samples tested from suspected cattle for LSDV, 106 were found positive. Of these, nine representative scab samples were further genetically characterized. Phylogenetic analysis based on the P32 and EEV gene sequences depicted that the LSDV strains of the present study had nucleotide identity of 100% and 99.10-99.45%, respectively with the LSDV/2019 field strains of India. Multiple sequence alignment of the EEV glycoprotein gene sequences revealed nucleotide polymorphisms at three positions viz., G178A, G253A and A459G in circulating LSDV/2022 field strains. Based on the median joining network analysis of the EEV gene, 12 haplotypes were identified among the LSDV populations. Population structure analysis corresponding to the EEV gene revealed high haplotype (0.8486 ± 0.026) and low nucleotide diversities (0.00636 ± 0.0006) and negative values for neutrality indices, indicating a high number of closely related haplotypes and the studied population may have undergone a recent expansion. The findings will help in understanding the distribution and dynamics of LSDV populations, which will prove pivotal in designing and implementing effective management and control strategies.

Evaluation of the immune responses in buffaloes vaccinated with a live-attenuated lumpy skin disease vaccine (Lumpi-ProVacInd).

Since 2019, Lumpy skin disease (LSD) has suddenly spread in many Asian countries, including India. LSD primarily occurs in cattle. However, recent LSD outbreaks in India have also revealed significant morbidity and production losses in buffaloes. This has raised concerns about the role of buffaloes in the epidemiology and transmission of LSD and necessitates the inclusion of buffaloes in the mass vaccination program for the prevention and control of the disease in the country. However, there is no significant data on the immune response in buffaloes following vaccination with the LSD vaccine. In this study, we evaluated antibody- and cell-mediated immune responses following vaccination with a newly developed live-attenuated LSD vaccine (Lumpi-ProVacInd). The detectable amount of anti-LSDV antibodies was observed at 1-2 months following vaccination, with a peak antibody titer at 3 months. Upon stimulation of the peripheral blood mononuclear cells (PBMCs) with the UV-inactivated LSDV antigen, there was a significant increase in CD8 + T cell counts in vaccinated animals as compared to the unvaccinated animals. Besides, vaccinated animals also showed a significant increase in IFN-γ levels upon antigenic stimulation of their PBMCs with LSDV antigen. In conclusion, the buffaloes also mount a potent antibody- and cell-mediated immune response following vaccination with Lumpi-ProVacInd.