Lumpy skin disease virus isolation, experimental infection, and evaluation of disease development in a calf.

Lumpy skin disease (LSD) is one of the most economically significant viral diseases of cattle caused by the Lumpy Skin Disease Virus (LSDV), classified as a member of the genus Capripoxvirus and belongs to the family Poxviridae. Nodular skin samples were collected from clinically sick cattle in the districts of Amuru and Wara Jarso Ethiopia to isolate LSD virus. The virus was isolated using primary lamb testis and kidney cells. The isolated LSDV was infected into a healthy calf while maintaining the necessary biosecurity measures to generate skin lesions and to assess disease progression using postmortem examinations. On the fourth day after virus inoculation, the calf developed typical LSD skin nodules with increased rectal temperature, which lasted until the 12th day, when they began to decrease. Viral shedding was detected in nasal, oral, and conjunctival swabs from 6 to 14 days after infection using real-time PCR. Post-mortem tissue specimens tested positive for LSD virus using real-time PCR and virus isolation. This study showed that LSDV were responsible for the LSD outbreaks, and the appearance of typical skin nodules accompanied by fever (> 39.5 °C) defined the virus's virulent status. The experimental infection with the isolated infectious LSDV could serve as a platform for future vaccine evaluation study using an LSDV challenge model.

A new variant of lumpy skin disease virus circulating in Vietnam based on sequencing analysis of GPCR gene.

Background: In 2021, Vietnam experienced an outbreak of Lumpy skin disease (LSD), which infected 207,687 cattle and buffaloes, as officially reported, and resulted in the culling of 29,182 animals. Aim: In this study, samples from cattle that died and showed typical signs of LSD in the Ha Tinh province of Vietnam were confirmed by three World Organization for Animal Health (WOAH)-recommended methods and further studied to compare the Vietnam and China reference strains to the new clinical cases. Methods: Three methods recommended by WOAH for agent detection (PCR, virus isolation, and transmission electron microscopy) were used to confirm this clinical LSD case. The sequence analysis of three well-known markers (P32, RPO30, and GPCR genes) has been utilized in Vietnam to understand this circulating pathogen better. Results: Our findings showed that the CX01 LSDV strain is 100% identical to the Vietnam reference strain HL01 and China reference strains based on P32 and RPO30 genes. Interestingly, analysis of the nucleotide sequence of the GPCR gene showed that the CX01 strain belongs to the same cluster as the reference strains, but it has branches different from those of both the HL01 and China LSDV strains. The nucleotide identification between the CX01 strain and these reference virus strains ranked 99.65%-99.91%, suggesting that it is a new variant of LSDV. Conclusion: This finding is new and indicates that at least two variants of the LSD virus were circulating in Vietnam based on analysis of the GPCR gene. Additionally, these results suggest that the sequence analysis of the GPCR gene is a great tool for subgrouping LSDV circulating in Vietnam.

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.

Design and assessment of a double antigen indirect ELISA for lumpy skin disease surveillance in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus of the genus Capripoxvirus, is rapidly emerging across most countries in Asia. Recently, LSD has been linked to very high morbidity and mortality rates. Until 2019, India remained free of LSD, resulting in a lack of locally developed diagnostic kits, biologicals, and other tools necessary for managing the disease in a country with such a large livestock population. Therefore, this study aimed to design and validate an indigenous and cost-effective in-house ELISA for large-scale screening of cattle samples for antibodies to LSDV. The viral major open reading frames ORF 095 and ORF 103 encoding virion core proteins were expressed in a prokaryotic system and the recombinant antigen cocktail was used for optimization and validation of an indirect ELISA (iELISA). The calculated relative diagnostic sensitivity and diagnostic specificity of the iELISA were 96.6 % and 95.1 %, respectively at the cut-off percent positivity (PP≥50 %). The in-house designed double-antigen iELISA was found effective to investigate the seroprevalence of LSDV in various geographical regions of India.

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.

Deciphering the genetic landscape of lumpy skin disease: Unraveling variable virulence through comprehensive genome sequence analysis in India.

Lumpy Skin Disease (LSD), a poxvirus disease affecting cattle, emerged in India in 2019 and intensified in 2022, resulting in significant economic losses for dairy farmers. There was unusual shift in mortality and morbidity patterns during the second wave. A comprehensive genetic study conducted, analyzing samples from 2019 to 2022 revealed circulation of two distinct subclades (subclade 1.2a and 1.2b) in India, with the latter showing a different pattern in morbidity and mortality. Notably, the Ankyrin repeats gene-based analysis could differentiate animals with varying clinical scores. Genetic variations were significant, with unique deletions identified, including a 12-nucleotide deletion in the GPCR gene in virus isolates collected during 2022 outbreaks, not reported earlier in Indian LSDV strains. A crucial finding was a significant 95-nucleotide deletion in the Functional Resolution Sequence (FRS) repeats of LSDV genomes from 2022 outbreaks, absent in 2019 samples. These deletions may have influenced the virus's virulence in India.

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.

Infection, dissemination, and transmission of lumpy skin disease virus in Aedes aegypti (Linnaeus), Culex tritaeniorhynchus (Giles), and Culex quinquefasciatus (Say) mosquitoes.

Lumpy skin disease virus (LSDV) is a transboundary viral disease in cattle and water buffaloes. Although this Poxvirus is supposedly transmitted by mechanical vectors, only a few studies have investigated the role of local vectors in the transmission of LSDV. This study examined the infection, dissemination, and transmission rates of LSDV in Aedes aegypti, Culex tritaeniorhynchus, and Culex quinquefasciatus following artificial membrane feeding of 102.7, 103.7, 104.7 TCID50/mL LSDV in sheep blood. The results demonstrated that these mosquito species were susceptible to LSDV, with Cx tritaeniorhynchus exhibiting significantly different characteristics from Ae. aegypti and Cx. quinquefasciatus. These three mosquito species were susceptible to LSDV. Ae. aegypti showed it as early as 2 days post-infection (dpi), indicating swift dissemination in this particular species. The extrinsic incubation period (EIP) of LSDV in Cx. tritaeniorhynchus and Cx. quinquefasciatus was 8 and 14 dpi, respectively. Ingestion of different viral titers in blood did not affect the infection, dissemination, or transmission rates of Cx. tritaeniorhynchus and Cx. quinquefasciatus. All rates remained consistently high at 8-14 dpi for Cx. tritaeniorhynchus. In all three species, LSDV remained detectable until 14 dpi. The present findings indicate that, Ae. aegypti, Cx. tritaeniorhynchus, and Cx. quinquefasciatus may act as vectors during the LSDV outbreak; their involvement may extend beyond being solely mechanical vectors.

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.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

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.

Lumpy skin disease outbreaks in vietnam, 2020.

Lumpy skin disease (LSD) is a transboundary, systemic, viral disease of cattle. The first outbreaks of LSD were reported in Lang Son Province of Vietnam (bordered to China), and an official document has been submitted to OIE on 1 November 2020. Here, we described first the genetic profiles of this pathogen based on four well-known marker regions. The LSD virus isolated in these first outbreaks was 100% identical to viruses isolated in China (2019) based on the p32 and RP030 genes. Additionally, it is very close to the virus isolated in Russia (2017) based on the p32, RP030, thymidine kinase and ORF103 genes (100%, 99.01%, 99.08% and 99.47% identities). This finding is new, and a success in LSD virus isolation using MDBK cells from first outbreaks is important for vaccine development to control and eradicate LSD in Vietnam.

Retention of lumpy skin disease virus in Stomoxys spp (Stomoxys calcitrans, Stomoxys sitiens, Stomoxys indica) following intrathoracic inoculation, Diptera: Muscidae.

Lumpy skin disease (LSD) is an emerging disease of cattle in Kazakhstan and the means of transmission remains uncertain. In the current study, retention of Lumpy Skin Disease Virus (LSDV) by three Stomoxys species following intrathoracic inoculation was demonstrated under laboratory conditions. A virulent LSDV strain was injected into the thorax of flies to bypass the midgut barrier. The fate of the pathogen in the hemolymph of the flies was examined using PCR and virus isolation tests. LSDV was isolated from all three Stomoxys species up to 24h post inoculation while virus DNA was detectable up to 7d post inoculation.

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.

Isolation and characterization of lumpy skin disease virus from cattle in India.

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells. Infection of the isolated LSDV to Vero cells did not produce cytopathic effect (CPE) until the 4th blind passage, but upon adaptation, it produced high viral titres in the cultured cells. The kinetics of viral DNA synthesis and one-step growth curve analysis suggested that Vero cell-adapted LSDV initiates synthesizing its genome at ~24 hours post-infection (hpi) with a peak level at ~96 hpi whereas evidence of progeny virus particles was observed at 36-48 hours (h) with a peak titre at ~120 h. To the best of our knowledge, this study describes the first successful isolation of LSDV in India, besides providing insights into the life cycle Vero cell-adapted LSDV.

Full-length genome characterization of a novel recombinant vaccine-like lumpy skin disease virus strain detected during the climatic winter in Russia, 2019.

An uncharacteristic outbreak of lumpy skin disease was reported in the Republic of Udmurtiya, Russia, during the climatic winter of March 2019. The causative lumpy skin disease virus (LSDV_Udmurtiya_Russia_2019) was shown to be a recombinant composed of a live attenuated Neethling-type vaccine strain as the dominant parental strain and a Kenyan KSGP/NI-2490-like virus as its minor parental strain, with 24 statistically significant recombination events that are not identical to those in LSDV Saratov/2017, in which 27 events were identified.

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes.

Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease.

Vaccination against lumpy skin disease (LSD) is crucial for maintaining the health of animals and the economic sustainability of farming. Either homologous vaccines consisting of live attenuated LSD virus (LSDV) or heterologous vaccines consisting of live attenuated sheeppox or goatpox virus (SPPV/GPPV) can be used for control of LSDV. Although SPPV/GTPV-based vaccines exhibit slightly lower efficacy than live attenuated LSDV vaccines, they do not cause vaccine-induced viremia, fever, and clinical symptoms of the disease following vaccination, caused by the replication capacity of live attenuated LSDVs. Recombination of capripoxviruses in the field was a long-standing hypothesis until a naturally occurring recombinant LSDV vaccine isolate was detected in Russia, where the sheeppox vaccine alone is used. This occurred after the initiation of vaccination campaigns using LSDV vaccines in the neighboring countries in 2017, when the first cases of presumed vaccine-like isolate circulation were documented with concurrent detection of a recombinant vaccine isolate in the field. The follow-up findings presented herein show that during the period from 2015 to 2018, the molecular epidemiology of LSDV in Russia split into two independent waves. The 2015-2016 epidemic was attributable to the field isolate. Whereas the 2017 epidemic and, in particular, the 2018 epidemic represented novel disease importations that were not genetically linked to the 2015-2016 field-type incursions. This demonstrated a new emergence rather than the continuation of the field-type epidemic. Since recombinant vaccine-like LSDV isolates appear to have entrenched across the country's border, the policy of using certain live vaccines requires revision in the context of the biosafety threat it presents.

Lumpy Skin Disease Is Characterized by Severe Multifocal Dermatitis With Necrotizing Fibrinoid Vasculitis Following Experimental Infection.

Lumpy skin disease is a high-consequence disease in cattle caused by infection with the poxvirus lumpy skin disease virus (LSDV). The virus is endemic in most countries in Africa and an emerging threat to cattle populations in Europe and Asia. As LSDV spreads into new regions, it is important that signs of disease are recognized promptly by animal caregivers. This study describes the gross, microscopic, and ultrastructural changes that occur over time in cattle experimentally challenged with LSDV. Four calves were inoculated with wildtype LSDV and monitored for 19 to 21 days. At 7 days after inoculation, 2 of the 4 cattle developed multifocal cutaneous nodules characteristic of LSD. Some lesions displayed a targetoid appearance. Histologically, intercellular and intracellular edema was present in the epidermis of some nodules. Occasional intracytoplasmic inclusion bodies were identified in keratinocytes. More severe and consistent changes were present in the dermis, with marked histiocytic inflammation and necrotizing fibrinoid vasculitis of dermal vessels, particularly the deep dermal plexus. Chronic lesions consisted of full-thickness necrosis of the dermis and epidermis. Lesions in other body organs were not a major feature of LSD in this study, highlighting the strong cutaneous tropism of this virus. Immunohistochemistry and electron microscopy identified LSDV-infected histiocytes and fibroblasts in the skin nodules of affected cattle. This study highlights the noteworthy lesions of LSDV and how they develop over time.