Molecular evidence of prolonged shedding of lumpy skin disease virus (LSDV) in the semen of naturally infected cattle bulls.

Lumpy skin disease (LSD) has emerged as a threat to cattle production in Asia, and India has been facing LSD epidemics since 2019. Although water buffalo (Bubalus bubalis) is susceptible to natural LSDV infection, there have been no confirmed reports of LSDV infection in water buffalo in India. In this study, we investigated suspected cases of LSD in water buffaloes from 12 Indian states and one union territory during 2020-2023. Buffaloes showed mild to moderate clinical disease with fever and nodular skin lesions, but most remained asymptomatic. Eighteen of 177 (10.18%) buffaloes in 12 districts in three states tested positive for LSDV by real-time PCR, while 22 of 57 (38.59%) from nine districts in six states tested positive for LSDV-neutralizing antibodies, demonstrating the prevalence of LSDV infection in buffaloes over a wider geographic area. Successful virus isolation and nucleotide sequencing confirmed natural LSDV infection in buffaloes. Phylogenetic analysis of complete GPCR, RPO30, and EEV gene sequences revealed the presence of wild-type strains of two divergent LSDV lineages (1.2.1 and 1.2.2) in buffaloes. The 1.2.2 strains were closely related to the dominant LSDV strain (subcluster 1.2.2, KSGP-like) circulating in India, while the 1.2.1 strains clustered with strains from the Middle East, Europe, and the Balkans, confirming that there have been multiple introductions of LSDV into India. The detection of viruses with identical sequences in buffaloes and local cattle implied that the LSDV strain found in buffaloes is probably of cattle origin. We also found evidence of cocirculation of LSDV 1.2.1 and 1.2.2 wild-type strains in the same area, highlighting the importance of LSDV surveillance and genetic analysis. This is the first confirmed report of natural LSDV infection in water buffaloes in India. Further investigations are needed to assess the impact of LSDV infection in buffalo production and the role of buffalo in LSD epidemiology.

Lumpy skin disease virus (LSDV) infection poses a significant threat to global cattle farming. Currently, effective therapeutic agents are lacking. TMP269, a small molecule inhibitor of class IIa histone deacetylase inhibitor, plays a vital role in cancer therapy. In this study, we demonstrated that TMP269 treatment inhibits the early-stage replication of LSDV in a dose-dependent manner. RNA sequencing data revealed that metabolism-related signaling pathways were significantly enriched after LSDV infection. Furthermore, untargeted metabolomics analysis revealed that lysophosphatidic acid (LPA), a key metabolite of the glycerophospholipid pathway, was upregulated following LSDV infection and downregulated after TMP269 treatment. In addition, exogenous LPA promotes LSDV replication by activating the mitogen-activated protein kinase (MEK)/extracellular-signal-regulated kinase (ERK) signaling pathway and suppressing the host's innate immune response. Furthermore, treatment with the LPA receptor inhibitor Ki16425 suppressed LSDV replication and promoted the host's innate immune response. These findings suggest that LSDV infection can induce LPA expression and aid viral activation of the MEK/ERK signaling pathway and escape of the host's innate immune response, whereas TMP269 treatment can inhibit LPA production and limit its promotion of LSDV replication. These data identified the antiviral mechanism of TMP269 and a novel mechanism by which LSDV inhibits host innate immune responses, providing insights into the development of new preventive or therapeutic strategies targeting altered metabolic pathways.IMPORTANCELumpy skin disease virus (LSDV) poses a significant threat to global cattle farming. Owing to insufficient research on LSDV infection, pathogenesis, and immune escape mechanisms, prevention and control methods against LSDV infection are lacking. Here, we found that TMP269, a class IIa histone deacetylase inhibitor, significantly inhibited LSDV replication. We further demonstrated that TMP269 altered LSDV infection-induced host glycerophospholipid metabolism. In addition, TMP269 decreased the accumulation of lysophosphatidic acid (LPA), a key metabolite in glycerophospholipid metabolism, induced by LSDV infection, and exogenous LPA-promoted LSDV replication by activating the mitogen-activated protein kinase (MEK)/extracellular-signal-regulated kinase (ERK) signaling pathway and suppressing the host innate immune response. Our findings identified the antiviral mechanism of TMP269 and a novel mechanism by which LSDV manipulates host signaling pathways to promote its replication, offering insights into the development of novel antiviral agents against LSDV infection.

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.

Lumpy skin disease (LSD) is an endemic infectious disease of cattle in Egypt. This survey aimed to define the prevalence of clinical and sub-clinical LSD virus (LSDV) infection among cattle and investigate their contact with water buffaloes (Bubalus bubalis) in order to improve the understanding of LSD epidemiology. Cattle and buffalo were examined owing to the appearance of skin lesions. Because clinical signs were consistent with LSDV infection, samples from cattle in a non-grazing dairy farm (n = 450) were submitted for LSDV testing together with those from the in-contact buffaloes (n = 100). Results revealed that the intra-herd percentage of cattle infected with LSDV varied with the detection method. This ranged from 22.4% to 65.4% by virus isolation (VI) and polymerase chain reaction (PCR), respectively, in clinical cattle samples, compared to 0% and 10% by VI and PCR in non-clinical cases. Using the neutralising index (NI), LSDV antibodies were found in 100% (n = 100) of the tested cow’s sera (NI = > 2.0 and ≥ 3.0), whereas buffalo’s sera (n = 34) displayed little increase in antibody level (NI ≥ 1.5). None of the buffalo were positive for LSDV by VI and PCR. In addition, there were no significant differences in LSD prevalence among the cattle with regard to age and sex. In conclusion, the occurrence of LSD in cattle warrants a further epidemiological study of the spread of the disease in the area and adoption of control and prevention strategies. In addition, the PCR assay was confirmed to be useful in the diagnosis of LSDV and for wider epidemiological studies.

Lumpy skin disease virus (LSDV) causes a nodular dermatitis in cattle and has high economic consequences in affected areas. Detection of LSDV exposure mostly relies on the humoral immune response, while the cell-mediated immune (CMI) response, an important hallmark of the immune reaction to LSDV, is neglected. We collected samples during 3 weeks post-vaccination of cattle with a Neethling-based live attenuated vaccine (LAV) and during 4 weeks post-LSDV infection under experimental conditions to i) investigate the development of the CMI response, ii) optimize an interferon-gamma release assay (IGRA) by comparing two matrices (whole blood and PBMCs) and different stimuli, and iii) evaluate the usefulness of an IGRA for detection of infection and vaccination. The CMI response to the Neethling LAV was detectable in all animals from 10 days post-vaccination, and importantly, preceded the detection of antibodies. A uniform CMI response to infection was already detected at its plateau level at 7 days post-infection in all animals and also preceded antibody detection. Whole blood and PBMCs allowed efficient IFN-γ secretion, but the IFN-γ response was significantly higher in PBMCs. Heat-inactivated antigen proved to be the stimulus of choice for LSD IGRA, providing increased sensitivity of the test and allowing its performance under BSL2 conditions. Under several conditions, LSDV IGRA showed a specificity of up to 100%. The LSD IGRA could be a powerful immunoassay for early detection of LSD and post-vaccination monitoring, making it worthwhile to explore its diagnostic characteristics in more animals and under field conditions.IMPORTANCEThe immune reaction to lumpy skin disease virus (LSDV) infection or vaccination is currently assessed with serological tests prone to suboptimal sensitivity, long processing time, and the necessity of biosafety level (BSL) 3 laboratories. Furthermore, the delayed or absent seroconversion indicates a need for an alternative immunoassay detecting immune reactions to LSDV exposure applicable in BSL2 settings. Seeing the known importance of cell-mediated immune (CMI) response against poxvirus infections, we evaluated the suitability of the interferon-gamma release assay (IGRA) test for detection of LSDV infection and vaccination. IGRA allowed early detection of the CMI response to LSDV infection (within 7 days) and vaccination (within 10 days) with a Neethling-based live attenuated vaccine, and the CMI response preceded the detection of seroconversion. Whole blood and heat-inactivated antigen increased IGRA sensitivity, making it suitable for application in BSL2 laboratories. This assay overcomes the downsides of currently available immunoassays, and these results encourage its further evaluation.

Lumpy skin disease (LSD) caused by lumpy skin disease virus (LSDV) inflicts significant economic losses in cattle production with impact on livelihoods of smallholders. This study reports the first occurrence of LSD in cattle in India and analyses epidemiological and genetic characterization data from LSD outbreaks in five districts of Odisha state in August 2019. In all, 182 of 2,539 cattle were affected with an apparent morbidity rate of 7.1% and no mortality. Out of 102 samples from 60 LSD suspected and 17 asymptomatic in-contact cattle tested, 29.87% cattle were positive by capripoxvirus generic PCR and 37.66% were positive by LSDV real-time PCR. All the in-contact cattle tested were negative for LSDV. Among affected cattle, LSDV genome was detected more in scabs (79.16%) than blood (31.81%) and frozen bull semen (20.45%). Differential diagnosis by PCR was negative for pseudo-LSD, buffalopox, cowpox, pseudo-cowpox and bovine papular stomatitis. Five selected PCR and real-time PCR-positive LSDV DNA were sequenced in three genomic regions, P32 (LSDV074), F (LSDV117) and RPO30 (LSDV036). Phylogenetic analysis based on partial P32 and F gene sequences and complete RPO30 gene sequences showed that all the five Indian LSDV strains were identical and clustered with other field strains of LSDV circulating globally. However, the F and RPO30 gene sequence analyses revealed that Indian LSDV strains are genetically closer to the South African NI2490/KSGP-like strains than the strains detected in Europe, which was rather surprising. The present study established the existence of LSDV in India and involvement of LSDV field strains in the outbreaks. Additionally, we provided evidence of LSDV shedding in semen of naturally infected bulls. Further studies are required to determine the source of LSD introduction, extent of spread, modes of transmission and impact on dairy cattle production in India and effective control measures must be undertaken urgently.

This study was carried out to explore epidemiological and molecular features of lumpy skin disease virus (LSDV) in the Aegean, Central Anatolian and Mediterranean regions of Turkey, to evaluate the risk factors associated with LSDV infection and to investigate the financial impact of LSD and potential role of the Culicoides spp. in the transmission of LSDV. Samples were obtained from 611 cattle, each from different farms, and each clinically suspected to be infected with LSDV during the months of July 2014 and June 2015. Culicoides spp. were trapped from April to June 2015. Genetic characterization of the local LSDV field isolates was conducted by sequencing G-protein-coupled chemokine receptor gene segment. Real-time PCR high-resolution melting analysis was used for distinguishing each type of capripoxviruses. Viral DNA was detected in 448 of the 611 animals and Culicoides midges. Three hundred and ninety-three of the 448 affected farms were surveyed. The morbidity and mortality rates detected were 12.3% and 6.4%, respectively. Phylogenetic analysis showed that the field isolates in this study were clustered together with other Africa and Middle East isolates. Genotyping of isolates from infected cattle has revealed the presence of LSDV. A generalized mixed linear model showed that there were positive associations between LSDV infection, European breeds, small-sized family-type farms and nearness of farm to a lake. The financial cost of disease presence in surveyed cattle farms was estimated to be 72.75 GBP per head. The sequence analysis of the mitochondrial cytochrome oxidase subunit I gene showed that the species of Culicoides in LSDV-positive pools was Culicoides punctatus. Detection of LSDV in Culicoides punctatus suggests that it may have played a role in transmitting LSDV. Furthermore, movement of infected animals into disease-free areas increases the risk of the transmission of LSD. Control strategies for LSDV infection should include consideration of the risk factors identified in this study.

Lumpy skin disease virus strategically modulates autophagy to facilitate viral replication in MDBK cells.

The present study monitored reproductive and health aspects of local cows in Egypt following natural infection with Lumpy skin disease virus (LSDV). The study was carried out on 640 mature Egyptian native cows reared at Lower Egypt. Cows were clinically examined and genital organs were scanned by ultrasonography and ovarian function was recorded. Blood samples were collected for virus isolation and propagation on calf Kidney cells, electron microscopy (EM) and for PCR assay. Moreover, progesterone level (ELISA), protein electrophoretic pattern (Polyaccrylamide Gel Electrophoresis), immunoglobulin concentrations (Radial Immune Diffusion) and some trace element values (Atomic Absorption Spectrophotometry) were determined in the serum. Results indicated that LSDV had typical cytopathic effect and plaque. EM showed that size of LSDV size ranges from 300 – 350 nm with crescent or ovoid shape. PCR assay indicated the presence of a band at 192 bp which belonged to viral attachment protein encoding gene. Epidemiological studies revealed that 25.47% of the examined cows were positive for LSDV with obvious year, age and seasonal variations. LSDV infected cows showed fever, become completely of food with skin nodules. Serum progesterone level was low (P < 0.01) during both follicular and luteal phases of the estrous cycle and 92.64% of infected cows had inactive ovaries. Infected cows showed obvious (P < 0.01) low albumin and high g globulin values as well as low concentrations of copper and iron (P < 0.01) in their serum as compared to non infected cow-mates. It was concluded that PCR is a useful method for rapid diagnosis of LSDV. Cows infected with LSDV mostly suffering from cessation of ovarian activity mainly due to poor body condition. Key words: LSDV, Cow, Ovary, PCR, infection, immunoglobulin.

Lumpy skin disease (LSD), a high-impact disease of cattle and water buffalo, is a direct threat to the European cattle industry. The causative agent is the lumpy skin disease virus (LSDV), an enveloped dsDNA virus which belongs to the family Poxviridae. Affected cattle present multiple cutaneous nodules that are characteristic of a LSDV infection. The case fatality rate of LSD is low, however affected animals suffer substantial production losses including weight loss and reduced milk production. On a wider scale, LSD is a high consequence transboundary disease with mandated export restrictions imposed on affected countries leading to substantial economic costs. Currently, there is no cure for LSDV infected cattle. Hence, mass vaccination is an important component in minimizing the spread of LSDV. There are only a handful of commercially available live attenuated vaccines in the current market. These vaccines vary in efficacy, quality, and safety, which leaves some scope for further development. This improvement is hampered by a poor understanding of the immunology of LSDV, particularly the protective immune mechanisms. In this project, we are investigating the immune response to LSDV with a focus on characterising the cell-mediated immune response. Peripheral blood mononuclear cells from LSDV immunised/infected cattle and flow cytometry assays are used to detect the production of IFN-γ by CD4+T-helper cells and CD8+cytotoxic T cells in response to live LSDV stimulation. Our goal is to improve understanding of the immune response to LSDV in order to develop effective vaccines and control programs in the future.

Lumpy skin disease virus (LSDV) infection, accompanied by loss of hide quality, poor reproductive efficiency, consistent degenerative emaciation, and milk yield reduction of animals, causes severe economic implications in endemic zones. The heterologous attenuated goat pox (GTPV) vaccine (AV41 strain) was used in China to prevent LSDV infection. Only a few LSDV detection methods that distinguish LSDV from GTPV vaccine strains have been reported before. For simple, rapid, and specific detection of LSDV, the real-time recombinase polymerase amplification (RPA) method was established with the specific primers and probes designed according to the conserved regions of ORF132 gene sequences. The assay could be finished within 20 min at a constant temperature (39 °C). This method had a limit of detection (LOD) of 15 copies/μL for LSDV and no cross-reaction with the nucleic acids of goat pox virus, infectious bovine rhinotracheitis virus, Pasteurella multocida, and bovine healthy tissue. Furthermore, 43 clinical samples were detected by this method and the real-time PCR recommended by the World Organisation for Animal Health (WOAH), with a kappa value, was 0.94. These results demonstrated that the real-time RPA method for detecting LSDV developed in this study was characterized by high sensitivity and specificity, which has wide application value in the clinical diagnosis and detection of LSDV in China.

The aim of this study was to investigate the prevalence and distribution patterns of LSDV infections in the provinces of northern Turkey, and to detect the factors influencing the epidemiology of LSD virus infections (age, breed, season, climate, geography, population dynamic, animal movement), as well as to assess the diagnostic value of the sampled materials in the diagnosis of LSDV infections. Lumpy skin disease (LSD) is an economically important cattle disease. The disease is endemic in many African countries, but outbreaks have also been reported in Turkey and the Middle East. In this study, a total of 564 samples (skin, blood and lung) from different cattle breeds (Jersey, Holstein-Friesian, Anatolian Black, Simmental and Brown Swiss) (n=465) in the many herds suspected of lumpy skin disease virus (LSDV) infection as clinically and macroscopic pathologic remarks, housed in the 7 different provinces of northern Turkey, were used for gel based conventional polymerase chain reaction (PCR). LSDV nucleic acid was detected in 259 of 564 (45.92%) materials by PCR. According to the result of PCR, the LSDV infection was diagnosed in 54.62% (254/465) of the sampled animals. The diagnostic value of necropsy and clinical materials such as skin and lung were determined as more valuable diagnostic materials in the diagnosis of LSDV infection by PCR. Data showed that LSDV infection was widespread in the provinces of northern Turkey and that the prevalence of the infection in the region varies in accordance with factors such as geographical conditions (climate, season, location etc.) and the method of breeding. .

Lumpy skin disease (LSD) is an emerging poxviral disease of cattle caused by the Capripoxvirus lumpy skin disease virus (LSDV) which generates widespread cutaneous lesions in affected animals. LSD is recognised as a transboundary high consequence disease in Africa where it contributes to rural poverty and food insecurity. In 2015 LSDV spread to southeastern Europe and currently poses a threat to cattle in neighbouring regions. Previous research indicates that LSDV is most likely transmitted by insect vectors however details of transmission pathways are unclear. This study was designed to identify the risk of transmission of LSDV posed by different insect vectors. A bovine experimental model of LSD was established in the high containment facilities at The Pirbright Institute. Potential insect vectors (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) were fed on LSDV-infected cattle then incubated for up to eight days. Cattle and insects were regularly sampled to quantify LSDV present in different tissues and vector species. This data was then used to model the dynamics of LSDV infection and transmission. All four species of vector successfully acquired LSDV from infected cattle and maintained the virus up to eight days post feeding. The outputs of this research will now be used to design more effective LSD control programmes.

The comparative study revealed that the hTERT-CSF cell line was the most susceptible cell to the Lumpy skin disease virus infection among eleven cells

Lumpy skin disease (LSD) has emerged as a global threat to cattle health and production. Although India has been encountering regular episodes of LSD epidemics on the mainland since 2019, the Andaman and Nicobar Islands remained free of LSD until 2021. In this study, we investigated the first LSD outbreak on Great Nicobar Island in 2022 and examined the genetic characteristics of the LSDV strain associated with this outbreak. The morbidity and mortality rates in cattle were 38.29% and 1.89%, respectively. We screened 123 samples (whole blood, nasal swab, or skin tissue) from 111 cattle by LSDV real-time PCR and sera from 86 cattle by LSDV antibody ELISA, and the results confirmed LSDV infection. Subsequent nucleotide sequencing and phylogenetic analysis of five selected marker genes (GPCR, RPO30, P32, EEV, and B22R) revealed that the LSDV strain from Great Nicobar Island resembled cluster 2.5 LSDV recombinant strains from East and Southeast Asia but was distinct from wild-type LSDV strains (1.2.1, 1.2.2) circulating in mainland India, indicating an exotic source of introduction. Phylogenetic analysis using a concatenated sequence (GPCR-RPO30-P32-EEV-B22R) showed that both the wild-type and recombinant LSDV strains formed well-supported clusters, indicating that this type of analysis may be used as an alternative to whole-genome sequence analysis. We also found that determination of the nucleotide sequence of the C-terminal 717 bp of the B22R gene may be sufficient for reliable assignment of circulating LSDV isolates to a particular cluster. This is the first report of the detection of a recombinant LSDV strain in India, demonstrating the spread of cluster 2.5 recombinant LSDV further into South Asia. Our findings highlight the value of LSDV surveillance and genetic analysis for LSDV epidemiology, which may be helpful for developing effective control strategies.