Genetic Characterization of Lumpy Skin Disease Viruses Circulating in Lesotho Cattle.

Lumpy skin disease is one of the fast-spreading viral diseases of cattle and buffalo that can potentially cause severe economic impact. Lesotho experienced LSD for the first time in 1947 and episodes of outbreaks occurred throughout the decades. In this study, eighteen specimens were collected from LSD-clinically diseased cattle between 2020 and 2022 from Mafeteng, Leribe, Maseru, Berea, and Mohales' Hoek districts of Lesotho. A total of 11 DNA samples were analyzed by PCR and sequencing of the extracellular enveloped virus (EEV) glycoprotein, G-protein-coupled chemokine receptor (GPCR), 30 kDa RNA polymerase subunit (RPO30), and B22R genes. All nucleotide sequences of the above-mentioned genes confirmed that the PCR amplicons of clinical samples are truly LSDV, as they were identical to respective LSDV isolates on the NCBI GenBank. Two of the elevem samples were further characterized by whole-genome sequencing. The analysis, based on both CaPV marker genes and complete genome sequences, revealed that the LSDV isolates from Lesotho cluster with the NW-like LSDVs, which includes the commonly circulating LSDV field isolates from Africa, the Middle East, the Balkans, Turkey, and Eastern Europe.

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

Lumpy skin disease outbreaks in Egypt during 2017-2018 among sheeppox vaccinated cattle: Epidemiological, pathological, and molecular findings.

The General Organization of the Veterinary Services in Egypt has adopted a sheeppox vaccination policy to control lumpy skin disease (LSD) in cattle. Over the course of the last two years, recurrent outbreaks were reported, with animals showing severe clinical signs and consequentially higher fatalities than that of cases reported in previous LSD outbreaks. A total of 1050 cattle showing typical clinical signs suggestive of LSD were clinically and pathologically investigated during 2017-2018. Skin nodules were collected and lumpy skin disease virus (LSDV) was screened in collected skin samples using PCR for the RPO-30 gene. Furthermore, the entire P32 protein coding gene was sequenced. Histopathology and immunohistochemistry of the skin nodules were also conducted. The obtained results showed an overall mortality rate of 6.86%. LSDV was confirmed in all the examined nodules as evidenced by immunohistochemistry and positive PCR amplification of the RPO30 gene. Sequencing analysis of the P32 gene revealed a highly conserved nature and genetic stability of the LSDV. The results of the present study show that the current vaccination protocol was not effective for a multitude of reasons. These results also serve as evidence for a strong recommendation of an amendment of homologous vaccine use aside from a complete coverage of cattle populations in order to reduce the incidence of LSD among cattle population in Egypt.

Molecular characterization of lumpy skin disease virus in Iran (2014-2018).

Lumpy skin disease was first reported in the western provinces of Iran in 2014, and this was followed by several outbreaks throughout the country. In this study, 10 Iranian lumpy skin disease virus (LSDV) samples collected during the period of 2014-2018 were characterized by sequence analysis of the GPCR, LSDV142, and IL10LP genes. Sequence comparison of the respective genes revealed a close relationship between Iranian LSDV isolates and viruses from Asia and Europe. Interestingly, some nucleotide sequence diversity was also observed in the IL10LP genes of the Iranian field isolates.

Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains.

BACKGROUND: Lumpy skin disease (LSD) is a contagious viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD has recently spread in Asia following outbreaks in the Middle East and Europe. The disease emerged in Bangladesh in July 2019 in the Chattogram district, then rapidly spread throughout the entire country. We investigated six LSD outbreaks in Bangladesh to record the clinical signs and collect samples for diagnostic confirmation. Furthermore, we performed the molecular characterization of Bangladesh isolates, analyzing the full RPO30 and GPCR genes and the partial EEV glycoprotein gene. RESULTS: Clinical observations revealed common LSD clinical signs in the affected cattle. PCR and real-time PCR, showed the presence of the LSDV genome in samples from all six districts. Phylogenetic analysis and detailed inspection of multiple sequence alignments revealed that Bangladesh isolates differ from common LSDV field isolates encountered in Africa, the Middle East, and Europe, as well as newly emerged LSDV variants in Russia and China. Instead, they were closely related to LSDV KSGP-0240, LSDV NI2490, and LSDV Kenya. CONCLUSIONS: These results show the importance of continuous monitoring and characterization of circulating strains and the need to continually refine the strategies for differentiating vaccine strains from field viruses.

Lumpy skin disease (LSD) outbreaks in cattle in Odisha state, India in August 2019: Epidemiological features and molecular studies.

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.

Emergence of a new lumpy skin disease virus variant in Kurgan Oblast, Russia, in 2018.

In this paper, we report the resurgence of lumpy skin disease (LSD) in Kurgan Oblast, Russia, in 2018. The majority of the outbreaks were silent with no mortality and congregated within an area with a radius of about 30 km located 1-50 km away from the national border with Kazakhstan. Following primary molecular diagnosis, LSD virus (LSDV) isolates were analyzed using a panel of PCR assays targeting different genetic loci, namely, LSD008 (vaccine), LSDV126 (field), and GPCR (vaccine and field), for differentiation and genotype assignment. All isolates were positive for the vaccine genotype of GPCR and negative for the other field targets tested. A PCR assay with melt curve analysis utilizing LSD008, developed in this work, indicated that the strains melted with a profile similar to those of field strains. Surprisingly, sequence analysis of the RPO30 and GPCR genes aligned the Kurgan/2018 isolate with KSGP O-240 at the GPCR locus, but with Saratov/2017 at the RPO30 locus. The latter cluster forms an association with a sub-cluster of the field strains comprising the South African KSGP O-240 strain and NI-2490 strain. Due to these incongruent phylogenetic patterns, the sequences of three additional loci ORF19 (Kelch-like protein), ORF52 (putative transcriptional elongation factor), and ORF87 (mutT motif protein) were investigated. Phylogenetic analysis of these additional loci placed the strain Kurgan/2018 in either vaccine or field groups, strongly suggesting a novel recombinant profile. This is another piece of evidence exposing the potential for recombination in capripoxviruses and the ignored danger of using live homologous vaccines against LSD. The necessity to revise the PCR-based strategy differentiating infected from vaccinated animals is discussed. The potential scenarios of incursion and the contribution of the KSGP/NI-2490-like strain to the emergence of the recently identified vaccine-like recombinant are discussed.

Molecular detection and phylogenetic analysis of lumpy skin disease virus from outbreaks in Uganda 2017-2018.

BACKGROUND: Lumpy skin disease (LSD) is an infectious viral disease of cattle caused by a Capripoxvirus. LSD has substantial economic implications, with infection resulting in permanent damage to the skin of affected animals which lowers their commercial value. In Uganda, LSD is endemic and cases of the disease are frequently reported to government authorities. This study was undertaken to molecularly characterize lumpy skin disease virus (LSDV) strains that have been circulating in Uganda between 2017 and 2018. Secondly, the study aimed to determine the phylogenetic relatedness of Ugandan LSDV sequences with published sequences, available in GenBank. RESULTS: A total of 7 blood samples and 16 skin nodule biopsies were screened for LSDV using PCR to confirm presence of LSDV nucleic acids. PCR positive samples were then characterised by amplifying the GPCR gene. These amplified genes were sequenced and phylogenetic trees were constructed. Out of the 23 samples analysed, 15 were positive for LSDV by PCR (65.2%). The LSDV GPCR sequences analysed contained the unique signatures of LSDV (A11, T12, T34, S99, and P199) which further confirmed their identity. Sequence comparison with vaccine strains revealed a 12 bp deletion unique to Ugandan outbreak strains. Phylogenetic analysis indicated that the LSDV sequences from this study clustered closely with sequences from neighboring East African countries and with LSDV strains from recent outbreaks in Europe. It was noted that the sequence diversity amongst LSDV strains from Africa was higher than diversity from Eurasia. CONCLUSION: The LSDV strains circulating in Uganda were closely related with sequences from neighboring African countries and from Eurasia. Comparison of the GPCR gene showed that outbreak strains differed from vaccine strains. This information is necessary to understand LSDV molecular epidemiology and to contribute knowledge towards the development of control strategies by the Government of Uganda.

The complete genome sequence of the lumpy skin disease virus vaccine Herbivac LS reveals a mutation in the superoxide dismutase gene homolog.

The lumpy skin disease virus (LSDV) vaccine, Herbivac LS, batch 008, was sequenced and found to differ from the Neethling vaccine strain in the locus encoding a superoxide dismutase (SOD) homolog. The presence of a SOD homolog, be it full-length (as in Herbivac LS) or truncated (as in Neethling) may affect vaccine immunogenicity.

Experimental lumpy skin disease virus infection of cattle: comparison of a field strain and a vaccine strain.

Lumpy skin disease virus (LSDV) infections can cause massive clinical signs in cattle and have great economic impact due to severe trade restrictions. For LSDV control, only live attenuated vaccines are commercially available, but they currently are not authorized in the European Union. Moreover, these vaccine virus strains can induce substantial side effects with clinical signs similar to infections with virulent LSDV. In our study, we compared clinical symptoms, viremia, and seroconversion of cattle inoculated either with a virulent field strain from North Macedonia isolated from diseased cattle in 2016 or with the attenuated LSDV vaccine strain "Neethling". Using specimens from the field and from experimental inoculation, different diagnostic tools, including a pan-capripox real-time qPCR, newly developed duplex real-time qPCR assays for differentiation between virulent and attenuated LSDV strains, and several serological methods (ELISA, indirect immunofluorescence test and serum neutralization test [SNT]) were evaluated. Our data show a high analytical sensitivity of both tested duplex real-time qPCR systems for the reliable distinction of LSDV field and vaccine strains. Moreover, the commercially available capripox double-antigen ELISA seems to be as specific as the SNT and therefore provides an excellent tool for rapid and simple serological examination of LSDV-vaccinated or infected cattle.

Detection of vaccine-like strains of lumpy skin disease virus in outbreaks in Russia in 2017.

Lumpy skin disease (LSD) has affected many regions of Russia since its first occurrence in 2015. The most devastating year for Russia was 2016, when the virus resurged following a modified stamping-out campaign, causing 313 outbreaks in 16 regions. To avoid unwanted adverse reactions following the use of live attenuated vaccines against LSD virus (LSDV), sheeppox-based vaccines were administered during vaccination campaigns. As a result, LSD was successfully contained in all Russian regions in 2017. In the same year, however, LSD emerged anew in a few regions of the Privolzhsky Federal District of Russia along the northern border of Kazakhstan, which then necessitated vaccinating cattle with a live attenuated LSDV vaccine. Although live attenuated LSDV vaccines are prohibited in Russia, several vaccine-like LSDV strains were identified in the 2017 outbreaks, including commercial farms and backyard animals exhibiting clinical signs consistent with those of field LSDV strains. Sequence alignments of three vaccine-like LSDV strains showed clear similarity to the corresponding RPO30 and GPCR gene sequences of commercial attenuated viruses. How vaccine-like strains spread into Russian cattle remains to be clarified.

Analysis and insights into recombination signals in lumpy skin disease virus recovered in the field.

Wide spread incidences of vaccine-like strains of lumpy skin disease virus (LSDV) have recently been reported in a Russian region with a neighboring country that actively vaccinate with a live attenuated LSD vaccine. The use of live-attenuated viruses (LAVs) as vaccines during an active outbreak, creates potential ground for coinfection of hosts and emergence of a strain combining genetic fragments of both parental vaccine and field strains. In this study, we analyse the vaccine-like strain LSDV RUSSIA/Saratov/2017 detected in Saratovskaya oblast, a region sharing border with Kazakhstan. To gain insight into possible recombination signals, a full-genome next-generation sequencing of the viral genome was performed using the Illumina platform. The genome contains the backbone of a live-attenuated vaccine with a patchwork of wild-type field virus DNA fragments located throughout. A total of 27 recombination events were identified. The average distance between the recombination sites was 3400 base pairs (bp). The impact of the recombination events on the virulence and transmission capacity of the identified virus remains to be clarified. These findings provide evidence for the first time of genetic exchanges between closely related strains of capripoxviruses in the field and a vaccine strain, and prompt a revisiting of the vaccination issue for a safe and efficacious prevention and control strategy of LSD.

Molecular characterization of lumpy skin disease virus in Namibia, 2017.

Between January and July 2017, lumpy skin disease (LSD) outbreaks were reported in cattle in Namibia. DNA was extracted from skin biopsies taken from 32 cattle, and the RNA polymerase 30 kDa subunit (RPO30) gene of the LSD virus (LSDV) was successfully amplified by PCR. Phylogenetic analysis revealed that the newly sequenced LSDV isolates from Namibia were identical to LSDV isolates identified previously in Burkina Faso, Egypt, Greece, Niger, Serbia and South Africa. Given that only unvaccinated herds were affected by LSD, it is recommended that the current vaccination programmes in Namibia be re-evaluated to allow nationwide coverage.

A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.

Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus.

Lumpy skin disease is of significant economic impact for the cattle industry in Africa. The disease is currently spreading aggressively in the Near East, posing a threat of incursion to Europe and Asia. Due to cross-protection within the Capripoxvirus genus, sheep pox virus (SPPV) vaccines have been widely used for cattle against lumpy skin disease virus (LSDV). In the Middle East and the Horn of Africa these vaccines have been associated with incomplete protection and adverse reactions in cattle post-vaccination. The present study confirms that the real identity of the commonly used Kenyan sheep and goat pox vaccine virus (KSGP) O-240 is not SPPV but is actually LSDV. The low level attenuation of this virus is likely to be not sufficient for safe use in cattle, causing clinical disease in vaccinated animals. In addition, Isiolo and Kedong goat pox strains, capable of infecting sheep, goats and cattle are identified for potential use as broad-spectrum vaccine candidates against all capripox diseases.

Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV).

Lumpy skin disease (LSD) was and still is a constant threat to the State of Israel, since the first outbreaks in 1989 and in 2006-2007. Recently, another massive outbreak occurred, at the beginning of July 2012, in the northern part of Israel. An intensive vaccination campaign with a sheeppox-based vaccine was initiated, in addition to culling symptomatic animals in the dairy herds. In spite of this, there was a need to apply extra efforts to completely contain and control the spread of the disease by introducing for the first time in Israel a vaccine based on the Neethling vaccine virus strain. However, in case of appearance of LSD symptoms it was essential to be able to distinguish between cattle-carried virulent strain and the vaccine strain. This paper describes the development and utilization of a molecular assay that can differentiate between the virulent isolates from the vaccine strain. The system is based on 3 different tests; it was found that the vaccine strain carries 27 bases less than the virulent virus in the extracellular enveloped virions (EEV) gene. A temperature-gradient PCRs were done using primers which are identical to the vaccine strain but differ at the 3' end nucleotides to the virulent virus. PCR-RFLP was carried out on the presence of an MboI site unique to the vaccine strain. Thus, all three tests presented here are able to differentiate specifically between the two viral appearances.

The use of lumpy skin disease virus genome termini for detection and phylogenetic analysis.

During 2006 and 2007 there were two outbreaks of lumpy skin disease (LSD) in Israel. An LSD virus (LSDV)-specific PCR assay was developed that can detected specifically LSDV even though the number of tested LSDV isolates were limited. Full-length sheep pox and LSDV genome sequences were aligned to find non-homologous regions, which were then used for preparing specific primers, whose specificity was tested against several LSDV DNA isolates and the system could detect all the different isolates. Specificity was tested with sheep pox, ORF and other DNA viruses such as bovine herpes 4: the primers did not support amplification of the expected-size fragments, therefore the system appears to be a valuable tool for detecting specifically LSDV. The newly developed system was activated at the first report of a possible disease outbreak. It confirmed the clinical picture, and was introduced subsequently into routine diagnosis. Phylogenetic analyses of a 466-bp fragment next to the genome ends showed that this system can distinguish between: sheep pox, goat pox and LSD, and the results revealed that the Israeli isolates from 2006 and 2007 are in the same clad and essentially identical to Ismaeliya 1989, Nigeria 1996, Senegal 1997, Cameroon 1996, the Kenya NI-2490 isolate, and the South African LD virulent isolate. In contrast the original 1958 LW Neethling vaccine appeared to be in a separate clad, suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the viruses tested suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the tested viruses or during the process of attenuating the virus by succession of egg inoculations.

Lumpy skin disease in southern Africa: a review of the disease and aspects of control.

This article reviews some of the important aspects of lumpy skin disease (LSD) that may impact on its successful control. A resurgence of the disease in the last decade has highlighted some constraints of the Neethling strain vaccine, but there is no evidence of vaccine breakdowns owing to the presence of heterologous field strains. More research is needed on epidemiology and transmission of LSD in South Africa to formulate control measures.