Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Epidemiological exploration of fleas and molecular identification of flea-borne viruses in Egyptian small ruminants.

The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt's Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Lessons Learned from Active Clinical and Laboratory Surveillance during the Sheep Pox Virus Outbreak in Spain, 2022-2023.

In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.

Structural and sequence analysis of the RPO30 gene of sheeppox and goatpox viruses from India.

Sheeppox and goatpox are transboundary viral diseases of sheep and goats that cause significant economic losses to small and marginal farmers worldwide, including India. Members of the genus Capripoxvirus (CaPV), namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV), are antigenically similar, and species differentiation can only be accomplished using molecular approaches. The present study aimed to understand the molecular epidemiology and host specificity of SPPV and GTPV circulating in India through sequencing and structural analysis of the RNA polymerase subunit-30 kDa (RPO30) gene. A total of 29 field isolates from sheep (n = 19) and goats (n = 10) belonging to different geographical regions of India during the period: Year 2015 to 2023, were analyzed based on the sequence and structure of the full-length RPO30 gene/protein. Phylogenetically, all the CaPV isolates were separated into three major clusters: SPPV, GTPV, and LSDV. Multiple sequence alignment revealed a highly conserved RPO30 gene, with a stretch of 21 nucleotide deletion in all SPPV isolates. Additionally, the RPO30 gene of the Indian SPPV and GTPV isolates possessed several species-specific conserved signature residues/motifs that could act as genotyping markers. Secondary structure analysis of the RPO30 protein showed four α-helices, two loops, and three turns, similar to that of the E4L protein of vaccinia virus (VACV). All the isolates in the present study exhibited host preferences across different states of India. Therefore, in order to protect vulnerable small ruminants from poxviral infections, it is recommended to take into consideration a homologous vaccination strategy.

Targeted Whole Genome Sequencing of the Capripoxvirus Genome from Clinical Tissue Samples and Lyophilized Vaccine Batches.

Diseases caused by Capripoxviruses (CaPVs) are of great economic importance in sheep, goats, and cattle. Since CaPV strains are serologically indistinguishable and genetically highly homologous, typing of closely related strains can only be achieved by whole-genome sequencing. In this chapter, we describe a robust, cost-effective, and widely applicable protocol for reconstructing (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches in less than a week. Taking advantage of the genetic similarity of CaPVs, a set of pan-CaPVs long-range PCRs was developed that covers the entire genome with only a limited number of tiled amplicons. The resulting amplicons can be sequenced on all currently available high-throughput sequencing platforms. As an example, we have included a detailed protocol for performing nanopore sequencing and a pipeline for assembling the resulting tiled amplicon data.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

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

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

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

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

In Silico Analysis of Honey Bee Peptides as Potential Inhibitors of Capripoxvirus DNA-Directed RNA Polymerase.

The genus Capripoxvirus belongs to the Poxviridae family. The sheeppox, goatpox, and lumpy skin disease viruses are three species of this genus with 96% identity in their genomes. These are financially devastating viral infections among cattle, which cause a reduction in animal products and lead to a loss in livestock industries. In the current study, the phylogenetic analysis was carried out to reveal the evolutionary relationships of Capripoxvirus species (i.e., sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV)) with other viruses from the Poxviridae family with >96% query coverage to find the similarity index among all members. The three viruses (i.e., SPPV, GTPV, and LSDV) joined the clade of Capripoxvirus of the Poxviridae family in the phylogenetic tree and exhibited close evolutionary relationships. The multiple sequence alignment using ClustalOmega revealed significant variations in the protein sequences of the DNA-dependent RNA polymerase of SPPV, GTPV, and LSDV. The three-dimensional structures of five selected bee peptides and DNA-directed RNA polymerase of SPPV, GTPV, and LSDV were predicted using trRosetta and I-TASSER and used for molecular docking and simulation studies. The protein-protein docking was carried out using HADDOCK server to explore the antiviral activity of peptides as honey bee proteins against SPPV, GTPV, and LSDV. In total, five peptides were docked to DNA-directed RNA polymerase of these viruses. The peptides mellitin and secapin-1 displayed the lowest binding scores (-106.9 +/- 7.2 kcal/mol and -101.4 +/- 11.3 kcal/mol, respectively) and the best patterns with stable complexes. The molecular dynamics simulation indicated that the complex of protein DNA-dependent RNA polymerase and the peptide melittin stayed firmly connected and the peptide binding to the receptor protein was stable. The findings of this study provide the evidence of bee peptides as potent antimicrobial agents against sheeppox, goatpox, and lumpy skin disease viruses with no complexity.

A novel triplex real-time PCR assay for the differentiation of lumpy skin disease virus, goatpox virus, and sheeppox virus.

Introduction: Three members of Capripoxvirus (CaPV) genus, including lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV), are mentioned as notifiable forms by World Organization for Animal Health. These viruses have negatively impacted ruminant farming industry worldwide, causing great economic losses. Although SPPV and GTPV cause more severe clinical disease in only one animal species, they can transfer between sheep and goats. Both homologous and heterologous immunization strategies are used to protect animals against CaPVs. However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of CaPV infection. Therefore, we developed a novel triplex real-time PCR (qPCR) for the differentiation of LSDV, GTPV, and SPPV. Methods: Universal primers were designed to detect pan-CaPV sequences. Species-specific minor groove binder (MGB)-based probes were designed, which were labeled with FAM for LSDV, HEX for GTPV, and ROX for SPPV. The sensitivity, specificity, reproducibility, and ability of detecting mixed infections were evaluated for the triplex qPCR. Further, 226 clinical samples of the infection and negative controls were subjected to the triplex qPCR, and the results were verified using PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods for PRO30 gene. Results: The triplex qPCR could successfully distinguish LSDV, GTPV, and SPPV in one reaction, and the assay sensitivity was 5.41, 27.70, and 17.28 copies/µL, respectively. No cross-reactivity was observed with other viruses causing common ruminant diseases, including des petits ruminants virus, foot-and-mouth disease virus, bluetongue virus, ovine contagious pustular dermatitis virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea-mucosal disease virus. Inter-and intra-assay variabilities were < 2.5%. The results indicated that the triplex qPCR was highly specific, sensitive, and reproducible. Simulation experiments revealed that this assay could successfully distinguish two or three viruses in case of mixed infections without any cross-reaction. For clinical samples, the results were completely consistent with the results of PCR-RFLP and sequencing. This demonstrated that the assay was reliable for clinical application. Discussion: The triplex qPCR is a robust, rapid, and simple tool for identifying various types of CaPV as it can successfully distinguish LSDV, GTPV, and SPPV in one reaction. Furthermore, the assay can facilitate more accurate disease diagnosis and surveillance for better control of CaPV infection.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

Genetic characterization of sheep pox virus strains from outbreaks in Central Russia in 2018-2019.

This study investigates the phylogenomic relatedness between sheep pox viruses (SPPVs) circulating in Central Russia in 2018-2019 with the NISKHI vaccine strain used in the country, based on their complete genome sequences. The sheep pox outbreaks occurred 1 year apart in the adjacent regions of Tula and Moscow. Full genome sequences were generated by sequencing DNA directly obtained from Trizol-extracted scabs, using the DNBSEQ-400 platform (MGI Tech, China). Phylogenetic analysis indicated that the SPPV isolates from Russia clusters with previously published sequences from Srinagar in the Kashmir province of India in 2000 (SPPV-Srinagar strain) as well as SPPV A strain from Kazakhstan in 2000. The aforementioned cluster belonged to a sister clade containing the NISKHI vaccine strain, thus indicating that the recent outbreaks were not genetically linked to the widely used vaccine.

One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10-6 ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10-5 ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. KEY POINTS: • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.

An in-depth bioinformatic analysis of the novel recombinant lumpy skin disease virus strains: from unique patterns to established lineage.

BACKGROUND: Since the first description of lumpy skin disease virus (LSDV) in Africa in the 1920's, it has brazenly spread beyond Africa into the Middle East, Europe and most recently Asia. In 2017 the first atypical LSDV recombinant strain was reported in Russia, composed of both a live-attenuated Neethling vaccine strain and Kenyan vaccine strain. An increase in LSDV research enabled a public release of numerous full genome sequences of unique recombinant LSDV strains from Kazakhstan, Russia, China and Vietnam. Prior to the recombinant strain first described in China in 2019, every new recombinant strain was genetically unique and each of these recombinants clustered in a monophyletic lineage. In this work, we provide the complete genome sequences of two novel recombinant strains of LSDV from Russia and attempt to gain more insight into genomic composition of all the recombinant strains currently available. This analysis will provide new insight into the global molecular epidemiology of LSDV. RESULTS: By sequencing and analyzing two novel recombinant strains Khabarovsk/2020 and Tomsk/2020, this study investigates the differences and similarities of all five the available recombinant LSDV lineages from different countries based on the SNPs inherited from the aforementioned parental strains. A total of seven recombinant strains: LSDV/Russia/Saratov/2017, LSDV/Russia/Udmurtya/2019, LSDV/KZ-Kostanay/Kazakhstan/2018, LSDV/Russia/Tyumen/2019, LSDV/GD01/China/2020 Khabarovsk/2020 and Tomsk/2020 were examined. It was observed that strains isolated prior to 2020 were composed of unique combinations of open reading frames, whilst from 2020 onwards all circulating strains in Russia and South-Eastern Asia belonged to a single lineage radiating out in the region. The first representative of this lineage is LSDV/GD01/China/2020. Interestingly, the other four unique recombinant strains as well as the newly established lineage, exhibit consistent patterns of targeted selection pointing to regions constantly selected for during the recombination-driven processes. CONCLUSION: This study highlights the inexplicable emergence of novel recombinant strains to be unique introductions of sibling viruses, with the most recent recombinant lineage establishing as the dominant strain across the south eastern Asian countries as evidenced by full genome sequence data. Overall, these findings indicate that LSDVs are subjected to accelerated evolutionary changes due to recombination in the face of homologous live attenuated vaccines as well as the slow genetic drift commonly observed in capripoxviruses curculatign in the field with hardly any genetic changes over decades.

Seroprevalence of sheeppox and goatpox virus in Asia and African continent: A systematic review and meta-analysis (Scientometrics).

Background and Aim: Two endemic capripox infectious diseases, sheeppox (SP) and goatpox (GP) are common in Asia, Africa, and the Middle East. Sheep and goats, in general, are considered current assets of small and marginal farmers and have significant economic value in terms of meat, wool, and skin/hide production. Sheep and goat populations in India total 148.88 million and 74.26 million, respectively. Capripox caused US$ 2.3 million (Indian Rupee [INR] 105 million) in economic damages in Maharashtra (India) alone, and it took over 6 years for a flock to recover from the outbreak. The projected yearly loss at the national level is US$ 27.47 million (INR 1250 million). As a result, Capripox diseases put small and marginal farmers under much financial strain. The present study estimates the seroprevalence of SP and GP diseases in the Asian and African continents using systematic review and meta-analysis. The results of the study will help researchers and policymakers to understand the spatial and temporal distribution of the disease and its burden. In addition, the results are also helpful to design and implement location-specific prevention and eradication measures against these diseases. Materials and Methods: Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines of Cochran collaborations were used for systematic review and subsequently meta-analysis were used. The literature was collected from various databases. Initial search string resulted in more than nine thousand articles for the period 2000 to 2020 using the different combinations of keywords and Boolean operators (or not) asterisk* and quotation marks. Out of 9398 papers, 80 studies were chosen for complete test reviews and quality bias evaluation using the inclusion and exclusion criteria. Finally, 21 articles were used for the meta-analysis. The statistical study employed fixed effects and random effects models using R. Results: Seroprevalence of SP and GP was calculated using studies with a cumulative sample size of 4352, out of which sheep and goats' samples together contribute 48%, followed by sheep (32%) and goat (21%). The result of the meta-regression revealed that detection techniques had a significant impact on the overall effect size at 5% level (Qm=14.12). Subgroup analysis of polymerase chain reaction (PCR) test with samples was further grouped into two categories based on the median, and it revealed that 62% of samples used PCR as a detecting test followed by group-II. Conclusion: From the study, it is concluded that SP and GP diseases are highly prevalent; hence, effective vaccines, proper education to farmers through extension activity, and transboundary disease movement restriction are necessary for the control and eradication of the disease.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

Cloning Strategies for the Generation of Recombinant Capripoxvirus Through the Use of Screening and Selection Markers.

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is commonly used to generate capripoxvirus knockout viruses (KO), and is based on the targeting of a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. An alternative step for the removal of both the EGFP and gpt cassettes and an optional selection step using CRISPR technology are also described.

Inhibition of bovine and ovine capripoxviruses (Lumpy skin disease virus and Sheeppox virus) by ivermectin occurs at different stages of propagation in vitro.

Capripoxvirus diseases are listed as reportable diseases by World Organization for Animal Health (OIE). Lumpy skin disease virus (LSDV) and sheeppox virus (SPPV), which can only be distinguished by molecular analysis, cause moderately, severe, or sometimes fatal infections in cattle and sheep. Even though vaccines are the most effective way to control the infection, their effectiveness may decrease in some cases. Therefore, it is significant to explore antiviral drugs against these diseases along with the vaccine. This study aimed to investigate the antiviral efficiency of ivermectin (IVM) at different stages of in vitro replication of LSDV and SPPV. For this purpose, viral titers (TCID50/mL) of the viruses not treated with IVM (0.0 µM) and treated with non-cytotoxic concentrations of IVM (1.0 and 2.5 µM) were compared during a nine-day (216 h) post-infection period by viral titration assay. At 2.5 µM concentrations of IVM, the mean viral titer was significantly (P<0.05) reduced by approximately three logs for the replication stage of LSDV and SPPV. To evaluate the antiviral activity of IVM against LSDV and SPPV by treatment at the virus attachment and penetration stages, the titers of the virus either untreated or treated with 2,5 µM IVM were compared by virus titration assay. The number of infectious virions for LSDV and SPPV were decreased by 99.82% and 99.87% at the viral replication stage, 68.38% and 25.01% at the attachment stage, and 57.83% and 0.0% at the penetration stage, respectively. It was determined that ivermectin is statistically more effective on LSDV than SPPV at the virus attachment and penetration stages (P<0.05). This study found that the drug IVM can inhibit capripoxviruses, including LSDV and SPPV at various stages of the propagation. Moreover, this research predicted the in vitro antiviral ability of IVM against capripoxvirus infections for the first time.