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.

Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans.

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

Epidemiological Dynamics of Foot-and-Mouth Disease in the Horn of Africa: The Role of Virus Diversity and Animal Movement.

The Horn of Africa is a large area of arid and semi-arid land, holding about 10% of the global and 40% of the entire African livestock population. The region's livestock production system is mainly extensive and pastoralist. It faces countless problems, such as a shortage of pastures and watering points, poor access to veterinary services, and multiple endemic diseases like foot-and-mouth disease (FMD). Foot-and-mouth disease is one of the most economically important livestock diseases worldwide and is endemic in most developing countries. Within Africa, five of the seven serotypes of the FMD virus (FMDV) are described, but serotype C is not circulating anymore, a burden unseen anywhere in the world. The enormous genetic diversity of FMDV is favored by an error-prone RNA-dependent RNA polymerase, intra-typic and inter-typic recombination, as well as the quasi-species nature of the virus. This paper describes the epidemiological dynamics of foot-and-mouth disease in the Horn of Africa with regard to the serotypes and topotypes distribution of FMDV, the livestock production systems practiced, animal movement, the role of wildlife, and the epidemiological complexity of FMD. Within this review, outbreak investigation data and serological studies confirm the endemicity of the disease in the Horn of Africa. Multiple topotypes of FMDV are described in the literature as circulating in the region, with further evolution of virus diversity predicted. A large susceptible livestock population and the presence of wild ungulates are described as complicating the epidemiology of the disease. Further, the husbandry practices and legal and illegal trading of livestock and their products, coupled with poor biosecurity practices, are also reported to impact the spread of FMDV within and between countries in the region. The porosity of borders for pastoralist herders fuels the unregulated transboundary livestock trade. There are no systematic control strategies in the region except for sporadic vaccination with locally produced vaccines, while literature indicates that effective control measures should also consider virus diversity, livestock movements/biosecurity, transboundary trade, and the reduction of contact with wild, susceptible ungulates.

Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine.

Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.

Assessment of the control measures of the Category A diseases of the Animal Health Law: prohibitions in restricted zones and risk-mitigating treatments for products of animal origin and other materials.

EFSA received a mandate from the European Commission to assess the effectiveness of prohibitions of certain activities in restricted zones, and of certain risk mitigation treatments for products of animal origin and other materials with respect to diseases included in the Category A list in the Animal Health Law (Regulation (EU) 2016/429). This opinion belongs to a series of opinions where other disease-specific control measures have been assessed. In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of (i) prohibiting the movements of certain products, notably germinal products (semen, oocytes, embryos and hatching eggs), products of animal origin and animal by-products and feed of plant origin, hay and straw, and (ii) risk mitigation treatments for products of animal origin. In terms of semen, oocytes, embryos and hatching eggs, it was agreed that there was a lack of evidence particularly for embryos and oocytes reflected in a varying degree of uncertainty, whether these commodities could potentially contain the pathogen under consideration. The scenario assessed did not consider whether the presence of pathogen would lead to infection in the recipient animal. In terms of animal products, certain animal by-products and movement of feed of plant origin and straw, the assessment considered the ability of the commodity to transmit disease to another animal if exposed. For most pathogens, products were to some degree considered a risk, but lack of field evidence contributed to the uncertainty, particularly as potential exposure of ruminants to meat products is concerned. In terms of the risk mitigating treatments, recommendations have been made for several of these treatments, because the treatment description is not complete, the evidence is poor or inconclusive, or the evidence points to the treatment being ineffective.

Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence?

From 2017 to 2019, several vaccine-like recombinant strains of lumpy skin disease virus (LSDV) were discovered in Kazakhstan and neighbouring regions of Russia and China. Shortly before their emergence, the authorities in Kazakhstan launched a mass vaccination campaign with the Neethling-based Lumpivax vaccine. Since none of the other countries in the affected region had used a homologous LSDV vaccine, it was soon suspected that the Lumpivax vaccine was the cause of these unusual LSDV strains. In this study, we performed a genome-wide molecular analysis to investigate the composition of two Lumpivax vaccine batches and to establish a possible link between the vaccine and the recent outbreaks. Although labelled as a pure Neethling-based LSDV vaccine, the Lumpivax vaccine appears to be a complex mixture of multiple CaPVs. Using an iterative enrichment/assembly strategy, we obtained the complete genomes of a Neethling-like LSDV vaccine strain, a KSGP-like LSDV vaccine strain and a Sudan-like GTPV strain. The same analysis also revealed the presence of several recombinant LSDV strains that were (almost) identical to the recently described vaccine-like LSDV strains. Based on their InDel/SNP signatures, the vaccine-like recombinant strains can be divided into four groups. Each group has a distinct breakpoint pattern resulting from multiple recombination events, with the number of genetic exchanges ranging from 126 to 146. The enormous divergence of the recombinant strains suggests that they arose during seed production. The recent emergence of vaccine-like LSDV strains in large parts of Asia is, therefore, most likely the result of a spillover from animals vaccinated with the Lumpivax vaccine.

Orbivirus Screening from Imported Captive Oryx in the United Arab Emirates Stresses the Importance of Pre-Import and Transit Measures.

From 1975 to 2021, the United Arab Emirates (UAE) imported more than 1300 live Arabian oryxes (AOs) and scimitar-horned oryxes (SHOs) for conservation programs. The objective of this study was to estimate the prevalence of orbiviruses Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in AOs and SHOs from captive herds in the UAE. Between October 2014 and April 2015, 16 AOs and 13 SHOs originating from Texas (USA) and 195 out of about 4000 SHOs from two locations in the UAE were blood sampled to be tested by indirect enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays. Eight imported AOs (50% CI [24.7-75.4%]) and eight imported SHOs (61.5% CI [31.6-86.1%]) were found BTV seropositive, in contrast with three out of 195 SHOs (1.5% CI [0.3-4.4%]) from the Emirates. BTV-2 genome was detected in 6/16 of the Arabian Oryx, and amongst those, one out of six was seronegative. None of the tested samples was found positive for EHDV. Our results illustrate the wide local variation regarding BTV seroprevalence in domestic and wild ruminants in the Arabian Peninsula. These results stress the need for pre-import risk assessment when considering translocation of wild ruminant species susceptible to orbiviruses not only in the country of destination but also where transit happens.

Assessment of the control measures of category A diseases of the Animal Health Law: Infection with rinderpest virus (Rinderpest).

EFSA received a mandate from the European Commission to assess the effectiveness of control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases ('Animal Health Law'). This opinion belongs to a series of opinions where these control measures are assessed, with this opinion covering the assessment of control measures for rinderpest (RP), the only animal disease to have been globally eradicated. In this opinion, the AHAW Panel reviewed the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radius of the protection and surveillance zone, and the minimum length of time the measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere. The transmission kernels used for the assessment of the minimum radius of the protection and surveillance zones are shown. Several scenarios for which control measures had to be assessed were agreed prior to the assessment. Considering that RP has been eradicated globally, a re-emergence that is not stopped in its early phases could have a devastating impact on animal health and the economy. The panel concludes that no suitable strategies are available to entirely mitigate the risk associated with granting derogations from killing of animals in an affected establishment or for animal movements. Therefore, the panel recommends to not grant any derogations. The monitoring period of 21 days was assessed as effective, except for the hypothetical first re-emergence of RP, when lack of awareness and diagnostic capability may extend the time to detection. It was concluded that the protection and the surveillance zones would contain 90% and > 99%, respectively, of the infections from an affected establishment. Enlarging the protection zone to 4 km would contain the disease spread with 95% probability.

Assessment of the control measures for category A diseases of Animal Health Law: Lumpy Skin Disease.

EFSA received a mandate from the EC to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases ('Animal Health Law'). This opinion belongs to a series of opinions where these control measures are assessed, with this opinion covering the assessment of control measures for Lumpy Skin Disease (LSD). In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of: i) clinical and laboratory sampling procedures, ii) monitoring period and iii) the minimum radius of the protection and surveillance zones, and the minimum length of time that measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere; nonetheless, the transmission kernels used for the assessment of the minimum radius of the protection and surveillance zones are shown. Several scenarios for which these control measures had to be assessed were designed and agreed prior to the start of the assessment. The monitoring period was assessed as effective, and based on the transmission kernels available, it was concluded that the protection zone of 20 km radius and the surveillance zone of 50 km radius would comprise > 99% of the transmission from an affected establishment if transmission occurred. Recommendations provided for each of the assessed scenarios aim to support the European Commission in the drafting of further pieces of legislation, as well as for plausible ad hoc requests in relation to LSD.

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and widely applicable method that allows to generate (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches. A set of pan-CaPVs long-range PCRs spanning the entire genome was designed to generate PCR amplicons that can be sequenced on commonly used high-throughput sequencing platforms: MiSeq (Illumina), RSII (PacBio) and MinION (Oxford Nanopore Technologies). The robustness of the LR-PCR strategy was evaluated for all 3 members of CaPV directly from a variety of samples, including clinical samples (N = 7), vaccine batches (N = 6), and virus isolates (N = 2). The sequencing method described here allows to reconstruct (nearly) complete CaPV genomes in less than a week and will aid researchers studying closely-related CaPV strains worldwide.

Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam.

Lumpy skin disease virus (LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.

Assessment of the control measures of the category A diseases of Animal Health Law: sheep and goat pox.

EFSA received a mandate from the European Commission to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases ('Animal Health Law'). This opinion belongs to a series of opinions where these control measures will be assessed, with this opinion covering the assessment of control measures for sheep and goat pox. In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radii of the protection and surveillance zones, and the minimum length of time the measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere; nonetheless, the transmission kernels used for the assessment of the minimum radii of the protection and surveillance zones are shown. Several scenarios for which these control measures had to be assessed were designed and agreed prior to the start of the assessment. Different risk-based sampling procedures based on clinical visits and laboratory testing are assessed in case of outbreak suspicion, granting animal movements and for repopulation purposes. The monitoring period of 21 days was assessed as effective. The estimated probability of transmission beyond the protection zone of 3 km radius from an infectious establishment is 9.6% (95% CI: 3.1-25.8%) and 2.3% (95% CI: 1-5.5%) for the surveillance zone of 10 km radius. This may be considered sufficient to contain the disease spread (95% probability of containing transmission corresponds to 5.3 Km). To contain 99% of the spread, the radius should be increased to 19.4 km (95% CI: 9.8-26.8). This may increase the number of farms in the surveillance zone, since the area would increase fourfold.

Bluetongue Virus Infections in Cattle Herds of Manabí Province of Ecuador.

Bluetongue (BT) is a viral disease transmitted by Culicoides (Diptera: Ceratopogonidae) to domestic and wild ruminants. Infections in cattle are mainly subclinical, but severe necrotic and hemorrhagic illness and death may occur depending on the strain of the virus and other factors; cattle act as a reservoir for the virus. Although the Ecuadorian coast has climatic conditions that favor the presence of the vector, there are few serologic or virologic BTV studies available. Manabí is a coastal province in which livestock farming is mostly implemented in the northern part. We conducted two studies to assess, for the first time, the presence of active BTV infections in Manabí province. We collected 430 serum samples from 38 randomly selected farms between March and July 2019 to perform BTV competitive ELISA. In addition, six seropositive farms were selected to place eight sentinel BTV-naive calves. All these calves were blood sampled and the presence of BTV RNA and antibodies was tested for by RT-PCR and competitive ELISA, respectively, once a week for 6-8 weeks until seroconversion was evidenced. A high individual seroprevalence (99%) was obtained, and all investigated farms had BTV seropositive animals. All sentinel calves became BTV viremic and seroconverted. The first viremia appeared after 2-5 weeks from arrival at the farm; they seroconverted 1-3 weeks later. We demonstrate for the first time that there is a high level of BTV circulation north of Manabí, with active infections on these farms. Integrated control strategies such as hygienic measures on farms to reduce midge populations would be advisable for the owners as mitigation measures.

A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control.

Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 105.6 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.

Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

Review: Vaccines and Vaccination against Lumpy Skin Disease.

The geographical distribution of lumpy skin disease (LSD), an economically important cattle disease caused by a capripoxvirus, has reached an unprecedented extent. Vaccination is the only way to prevent the spread of the infection in endemic and newly affected regions. Yet, in the event of an outbreak, selection of the best vaccine is a major challenge for veterinary authorities and farmers. Decision makers need sound scientific information to support their decisions and subsequent actions. The available vaccine products vary in terms of quality, efficacy, safety, side effects, and price. The pros and cons of different types of live attenuated and inactivated vaccines, vaccination strategies, and associated risks are discussed. Seroconversion, which typically follows vaccination, places specific demands on the tools and methods used to evaluate the effectiveness of the LSD vaccination campaigns in the field. We aimed to give a comprehensive update on available vaccines and vaccination against LSD, to better prepare affected and at-risk countries to control LSD and ensure the safe trade of cattle.

The Importance of Quality Control of LSDV Live Attenuated Vaccines for Its Safe Application in the Field.

Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.

Comparative Evaluation of Lumpy Skin Disease Virus-Based Live Attenuated Vaccines.

Vaccines form the cornerstone of any control, eradication and preventative strategy and this is no different for lumpy skin disease. However, the usefulness of a vaccine is determined by a multiplicity of factors which include stability, efficiency, safety and ease of use, to name a few. Although the vaccination campaign in the Balkans against lumpy skin disease virus (LSDV) was successful and has been implemented with success in the past in other countries, data of vaccine failure have also been reported. It was therefore the purpose of this study to compare five homologous live attenuated LSDV vaccines (LSDV LAV) in a standardized setting. All five LSDV LAVs studied were able to protect against a challenge with virulent LSDV. Aside from small differences in serological responses, important differences were seen in side effects such as a local reaction and a Neethling response upon vaccination between the analyzed vaccines. These observations can have important implications in the applicability in the field for some of these LSDV LAVs.

Scientific Opinion on the assessment of the control measures for category A diseases of Animal Health Law: Foot and Mouth Disease.

EFSA received a mandate from the European Commission to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases ('Animal Health Law'). This opinion belongs to a series of opinions where these control measures will be assessed, with this opinion covering the assessment of control measures for foot and mouth disease (FMD). In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of: i) clinical and laboratory sampling procedures, ii) monitoring period and iii) the minimum radius of the protection and surveillance zones, and the minimum length of time the measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere; nonetheless, the transmission kernels used for the assessment of the minimum radius of the protection zone of 3 km and of the surveillance zone of 10 km are shown. Several scenarios for which these control measures had to be assessed were designed and agreed prior to the start of the assessment. The monitoring period of 21 days was assessed as effective, and it was concluded that the protection and the surveillance zones comprise > 99% of the infections from an affected establishment if transmission occurred. Recommendations, provided for each of the scenarios assessed, aim to support the European Commission in the drafting of further pieces of legislation, as well as for plausible ad hoc requests in relation to FMD.

Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg.

Bluetongue is one of the major diseases of ruminants listed by the World Organisation for Animal Health. Bluetongue virus serotype 8 (BTV-8) has been considered enzootic in France since 2018. Here, we report the nearly complete genome sequences of two BTV-8 isolates from the 2020 outbreak in the Grand Duchy of Luxembourg.