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.

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.

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.

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.

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.

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.

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.

A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus.

In this paper, we report on the development of a real time high-resolution melting (HRM) PCR assay for detection and differentiation among sheep pox virus (SPPV), goat pox virus (GTPV), field isolates and vaccine strains of lumpy skin disease virus (LSDV) based on high-resolution melting curve analysis of their target PCR amplicons. A 111 bp region of LSDV010 ORF, which harbors unique genetic differences for each of these viral species, was selected as the PCR target in this study. During the validation of this assay using DNA from clinical isolates originated from naturally infected animals from the different geographic locations and reference strains, the obtained PCR amplicons demonstrated that the melting temperature picks were specific for each tested viral species, i.e., 74.56 ±â€¯0.04 °C for field LSDV, 74.95 ±â€¯0.08 °C for vaccine LSDV, 74.24 ±â€¯0.06 °C for SPPV and 73.61 ±â€¯0.04 °C for GTPV. The assessment of the assay sensitivity utilizing a LSDV field strain as a PCR template revealed the assay detection limit as low as 0.1 TCD50 lg/ml. Overall, this assay based on Rotor-Gene Q (QIAGEN) platform was shown to be reproducible across replicates and operators and can be recommended as an additional diagnostic tool to the currently available molecular assays for detection and differentiation of the genus Capripoxvirus species, including the differentiation of vaccine strains of LSDV from field isolates. The assay can be used for detection of these viruses in animal- and insect-derived field specimens.

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.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

BACKGROUND: Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. RESULTS: The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. CONCLUSION: The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Lumpy skin disease in cattle in central Ethiopia: outbreak investigation and isolation and molecular detection of the virus.

The study was a combination of two investigations into active outbreaks of lumpy skin disease (LSD) in cattle in central Ethiopia and a retrospective analysis of outbreak reports between January 2007 and December 2011 covering the entire country. Active outbreaks were investigated in four districts of central Ethiopia: Adama, Wenji, Mojo and Welenchiti. A semi-structured questionnaire was used to acquire data at individual and herd levels, and tissue samples were collected for viral isolation and characterisation. The retrospective analyses showed that, during the five-year period, a total of 1,675 outbreaks were reported, with 62,176 cases and 4,372 deaths. The highest number of outbreaks was reported in Oromia (1,066), followed by Amhara (365) and the Southern Nations, Nationalities and People's Region (123). Outbreaks were more frequently observed between September and December and the highest number of outbreaks was reported in 2010. During the period studied, a total of 2,174 local zebu cattle were clinically examined and morbidity and mortality rates of 13.61% (296) and 4.97% (108) were recorded, respectively. Analysis of the active outbreaks revealed a relatively consistent morbidity rate, with the highest observed in Adama (15.38%), followed by Wenji (10.26%). The highest mortality rates were also observed in Adama (5.89%) and Wenji (3.42%). The LSD virus was isolated from 22 samples and all tested positive in polymerase chain reaction analysis. The disease was observed in the cattle regardless of previous vaccination with Kenyan sheep- and goat-pox vaccine; thus, vaccine efficacy was assessed under field conditions and the authors' findings, together with a possible remedy, are presented in this paper.

Evidence of lumpy skin disease virus over-wintering by transstadial persistence in Amblyomma hebraeum and transovarial persistence in Rhipicephalus decoloratus ticks.

Lumpy skin disease is a debilitating cattle disease caused by the lumpy skin disease virus (LSDV), belonging to the genus Capripoxvirus. Epidemics of the disease usually occur in summer, when insect activity is high. Limited information is available on how LSDV persists during inter-epidemic periods. Transmission of LSDV by mosquitoes such as Aedes aegypti has been shown to be mechanical, there is no carrier state in cattle and the role of wildlife in the epidemiology of the disease seems to be of minor importance. Recent studies in ticks have shown transstadial persistence of LSDV in Rhipicephalus appendiculatus and Amblyomma hebraeum as well as transovarial persistence of the virus in Rhipicephalus decoloratus, R. appendiculatus and A. hebraeum. The over-wintering of ticks off the host as part of their life cycles is well known: A. hebraeum and R. appendiculatus over-winter, for example, on the ground as engorged nymphs/unfed (emergent) adults while R. decoloratus over-winters on the ground as engorged females. In this study, transstadial and transovarial persistence of LSDV from experimentally infected A. hebraeum nymphs and R. decoloratus females after exposure to cold temperatures of 5 °C at night and 20 °C during the day for 2 months was reported. This observation suggests possible over-wintering of the virus in these tick species.

Transovarial passage and transmission of LSDV by Amblyomma hebraeum, Rhipicephalus appendiculatus and Rhipicephalus decoloratus.

Lumpy skin disease (LSD), an acute, sub-acute or inapparent disease of cattle, is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus in the family Poxviridae. LSD is characterised by high fever, formation of circumscribed skin lesions and ulcerative lesions on the mucous membranes of the mouth, respiratory and digestive tracts. It is an economically important disease due to the permanent damage to hides, the reduction in productivity and trade restrictions imposed on affected areas. Transmission has been associated with blood-feeding insects such as stable flies (Stomoxysis calcitrans) and mosquitoes (Aedes aegypti). Mechanical (intrastadial) and transstadial transmission by Amblyomma hebraeum and Rhipicephalus appendiculatus as well as transovarial transmission by R. decoloratus have been reported. In this study transovarial passage of LSDV to larvae and subsequent transmission to recipient animals were demonstrated. The finding of transovarial passage of LSDV in female ticks shows the potential for A. hebraeum, R. appendiculatus and R. decoloratus to be reservoir hosts for LSDV.

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.

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.

Characterization and molecular identification of the lumpy skin disease virus in cattle in the Mekong Delta of Vietnam.

Background: Lumpy skin disease (LSD) is caused by a virus belonging to the genus Capripoxvirus, exhibiting clinical symptoms ranging from mild signs to the development of nodules. LSD emerged in Asia and Southeast Asia, including Vietnam, in October 2020 and has since spread throughout the region, resulting in productivity and economic losses. Aim: This study aimed to investigate the virus-causing papular dermatitis in cattle from the Mekong Delta region of Vietnam by analyzing its GPCR gene and assessing its evolutionary relationship with sequences in the GenBank database. Methods: Blood samples (n = 180) were collected from cattle farms in Ben Tre, Tien Giang, and Tra Vinh provinces. PCR targeting the P32 antigen gene was utilized to detect LSDV presence, and GPCR gene amplification was performed to assess genetic variability. Results: LSDV was detected in 8.33% (15/180) of the samples using PCR targeting the P32 antigen gene. Each sample that tested positive for LSDV demonstrated complete amplification of the GPCR gene. Sequence alignments and phylogenetic analyses of the GPCR gene revealed that Mekong Delta LSDV isolates shared genetic similarities and possessed a 12-nucleotide insertion comparable to strains from China in 2019 and Northern Vietnam in 2020. Conclusion: This study provides preliminary insights into the molecular characteristics of LSDV in cattle from the Mekong Delta region of Vietnam. The observed genetic relatedness to other LSDV sequences from Asia and Southeast Asia underscores the importance of regional surveillance and control measures. These findings contribute to the development of effective strategies for LSDV control and prevention.

Protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach.

Lumpy skin disease (LSD) is a viral disease predominantly affecting cattle caused by a poxvirus belonging to the capripoxvirus genus. Here, we present a protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach. We describe steps for DNA extraction, viral DNA enrichment, amplicon pooling and purification, and library preparation and pooling. We then detail procedures for sequencing and computational analysis. This protocol can be adapted to any Illumina sequencing platform as an accelerated and scalable system. For complete details on the use and execution of this protocol, please refer to Bhatt et al.1,2.

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.