BACKGROUND: Direct whole genome sequencing of Capripox virus genomes from diagnostic samples is not always straightforward. Low viral content in a sample, biased sequencing and subsequent assembly and mapping methods may all influence the outcome. METHODS: In this study we have tested and compared six next generation sequencing approaches on a homogenized skin sample from a bull infected with LSDV. We compared enrichment vs. non-enrichment strategies, different library preparation methods, short read Illumina sequencing with long read sequencing methods. RESULTS: We found that methods that use an unbound transposon during tagmentation produced unbalanced results and lower target read yield versus methods that use other approaches to the tagmentation step. We further find that the use of hybrid capture probes increased the number of target reads. The result of subsequent mapping and assembly steps are influenced by the choice of reference when using reference-based assembly approaches. CONCLUSIONS: When using a short read sequencing approach we advise to use a transposon free method or a method with bound transposons for DNA fragmentation. These methods outperform kits that employ free transposons for DNA fragmentation when targeting AT-rich genomes. When mapping the reads it is best to use a reference for assembly that is as closely related as possible to the sample under study. Mapping problems can be resolved by long read sequencing which we recommend for denovo whole genome sequencing. Pacific Bioscience based long read sequencing outperforms Oxford Nanopore sequencing because it is less error prone. The ONT approach used, displays the same bias as the transposon based approach from Illumina and is therefore less suitable when attempting (Capri)pox whole genome sequencing.

In the original publication [...].

Abstract Lumpy skin disease virus (LSDV) is an emerging livestock capripoxvirus (CaPV) that continues to cause substantial economic losses across Africa, Asia and Europe. However, important uncertainties remain regarding LSDV genome structure, particularly at the telomeric inverted terminal repeats (ITRs) that are central to host interaction, replication and adaptive evolution. Structural variation at the ITRs is driven by recombination during virus replication. Previous assemblies have relied primarily on short-read sequencing, which provides limited resolution of repeat-rich telomeric regions and propagates structural ambiguities into downstream annotation and comparative analyses. Here, we present a high-quality telomere-to-telomere (T2T) assembly of the LSDV Oman 2009 isolate, generated using a hybrid approach that integrates short Illumina reads with higher-accuracy long Nanopore reads. The resulting 151,091 bp genome contains 157 annotated open reading frames and fully resolves complex repeat-rich structures at both ITRs. This new reference corrects misassemblies present in earlier LSDV genomes and confirms clade-specific gene truncations. This genome provides a robust foundation for improved genomic surveillance, accurate read mapping, mutation detection and evolutionary inference of LSDV, and demonstrates the value of long-read approaches for resolving complex CaPV genome structures.

Hong Kong veterinary authorities responded to a series of suspected outbreaks of African swine fever in domestic pigs in the New Territories of the Hong Kong Special Administrative Region from November 2023 to January 2024. Cases were identified on 10 farms, with one farm having a single pig with an inconclusive result. Subsequent genetic analysis suggested a more complicated picture than initially considered. Full genome sequencing identified a genotype II virus with a novel deletion in the left of the genome, as well as a genotype I/II hybrid virus similar to those previously reported in the Chinese mainland, Vietnam, and Russia. The replication kinetics of the two viruses were compared to reference strains in vitro, and the virulence of the genotype I/II hybrid was tested in vivo alongside an African genotype XV virus. Both viruses caused high fever and inappetence, with animals reaching severe humane endpoints within 6 days.IMPORTANCEAfrican swine fever virus causes a lethal hemorrhagic disease in domestic and wild pigs that is present in Africa, Asia, Europe, Oceania, and the Caribbean island of Hispaniola. The virus is a serious threat to pig farmers, global food security, and biodiversity. The virus has been circulating in Eurasia for nearly 20 years, and both small and large changes in the genome have been observed, including a new recombinant virus that emerged in 2021. Full genome sequencing of viruses is required to identify any changes that are important for understanding the epidemiology of the virus, as well as guiding vaccine design and selection. Here, we report genomic data from outbreaks in Hong Kong SAR during Winter 2023/2034 and evaluate the pathogenicity of a hybrid-recombinant virus. We show that minor changes to the hybrid-recombinant genome do not influence virulence and show that this virus, along with a previously uncharacterized isolate from Africa, can be used to test vaccine efficacy in future studies.

Bluetongue was historically a disease of the tropics and subtropics, but over the past 20 yr has emerged in temperate regions such as northern Europe. Multiple serotypes of bluetongue virus (BTV) have been introduced into northern Europe, with some becoming enzootic, most notably 3, 4, and 8 in France. This review describes the step-change in the occurrence of bluetongue across northern Europe with a focus on changes since the re-emergence of BTV-8 in France in 2015. Five countries in northern Europe are specifically discussed-Belgium, France, the Netherlands, the United Kingdom, and Switzerland. These countries have all been significantly affected by the widespread outbreak of BTV-3, which emerged in 2023. Here, the latest data on case numbers, disease and Culicoides surveillance, and the current state of play for each country are presented. The latest research into vector competence, transmission studies, and advances in vaccination and vector control, relevant to northern Europe, are summarized. Finally, climate change and globalization are critically examined as reasons for the epidemiological changes in BTV occurrence across Europe.

Lumpy skin disease virus (LSDV) is a poxvirus that can cause severe, systemic disease in cattle. By far the most important route of transmission of LSDV is mechanical transmission via haematophagous arthropod vectors. However we lack detailed information on this process including the likelihood of transmission by different vector species. This study used an experimental bovine model of LSDV transmission to quantify the transmission of LSDV from an infected donor to a naïve recipient calf. Three species of Diptera representing different vector groups were included ( Stomoxys calcitrans , Aedes aegypti and Culicoides nubeculosus, respectively a large biting fly, a mosquito and a midge), and the clinical, virological and immunological outcomes in the recipient calves studied. The ability of Ae. aegypti to mechanically transmit LSDV following feeding on an artificial membrane feeding system was also examined. Both Ae. aegypti and S. calcitrans were able to transmit LSDV, resulting in disease in recipient calves. Bites from virus-positive C. nubeculosus did not result in disease in recipient calves, though the presence of neutralising antibodies in these recipients indicated exposure to virus or virus components. Ae. aegypti successfully transmitted LSDV following feeding on LSDV-spiked blood through an artificial membrane feeding system, validating this laboratory model as a future replacement for donor cattle. Mathematical models of the data were generated and predicted S. calcitrans to be the most efficient vector of LSDV of the insects tested with a reproduction number (R­) of 5.8. Importance Lumpy skin disease virus (LSDV) is a neglected, rapidly emerging pathogen of cattle that has spread into Europe and throughout the Middle East and Asia over the past ten years. Lack of understanding of the mechanism of transmission of LSDV has hampered efforts to control its rapid spread. This study compares the ability of three model species of Diptera ( Stomoxys calcitrans , Aedes aegypti and Culicoides nubeculosus ) to mechanically transmit the virus to cattle, generating high quality quantitative data to facilitate mathematical modelling of virus transmission. S. calcitrans was identified as a potential driver of LSDV transmission with a R ­of 5.8. This work provides new insights into the vector transmission of LSDV that can be used to design more targeted and effective disease control programmes. ### Competing Interest Statement The authors have declared no competing interest. BBSRC, BB/R002606, BB/R008833, BB/T005173/1, BBS/E/I/0000733 BBS/E/I/0000739, BBS/E/I/0000736 BBS/E/I/0000738, BBS/E/I/0000737 MSD Animal Health European Union, 773701

This study represents the first report on the detection and whole-genome sequencing of African swine fever (ASF) viruses in wild boar in Hong Kong in 2021–2023. Wild boar samples collected via an ASF surveillance program by the Agriculture, Fisheries, and Conservation Department were tested for ASF viruses (ASFVs) using real-time polymerase chain reaction. ASF-positive carcasses were detected in four cases and hemadsorption, virus isolation, and whole-genome sequencing were conducted. The B646L gene, E183L gene, central variable region within the B602L gene, intergenic region between the I73R and I329L genes, EP420R gene, and multigene family members of the four ASFV strains were compared. The whole-genome phylogenetic relationships were studied. The comparative analysis of the genomes indicates that the ASFVs in these four cases have genetic similarities to Asian genotype II ASFVs, but are genetically distinct from each other, as well as the ASFV previously identified in a domestic pig farm in Hong Kong in 2021.

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organisation for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Agüero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH Reference Laboratory for AHS in the Republic of South Africa (RSA) reported an AHSV variant undetected by the Agüero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Agüero method was validated by the WOAH Reference Laboratories in Spain and the UK, employing a broad collection of AHSV strains and clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Agüero versus the WOAH reference rRT-PCRs showed that the modified method exhibited good diagnostic performance and enabled detection of the new RSA AHSV variant nucleic acid.

African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

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

African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4–6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide.

Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.

A recombinant, replication-defective, adenovirus-vectored vaccine expressing the H surface glycoprotein of peste des petits ruminants virus (PPRV) has previously been shown to protect goats from challenge with wild-type PPRV at up to 4 months post vaccination. Here, we present the results of a longer-term trial of the protection provided by such a vaccine, challenging animals at 6, 9, 12 and 15 months post vaccination. Vaccinated animals developed high levels of anti-PPRV H protein antibodies, which were virus-neutralising, and the level of these antibodies was maintained for the duration of the trial. The vaccinated animals were largely protected against overt clinical disease from the challenge virus. Although viral genome was intermittently detected in blood samples, nasal and/or ocular swabs of vaccinated goats post challenge, viral RNA levels were significantly lower compared to unvaccinated control animals and vaccinated goats did not appear to excrete live virus. This protection, like the antibody response, was maintained at the same level for at least 15 months after vaccination. In addition, we showed that animals that have been vaccinated with the adenovirus-based vaccine can be revaccinated with the same vaccine after 12 months and showed an increased anti-PPRV antibody response after this boost vaccination. Such vaccines, which provide a DIVA capability, would therefore be suitable for use when the current live attenuated PPRV vaccines are withdrawn at the end of the ongoing global PPR eradication campaign.

BACKGROUND:Bluetongue virus serotype 3 (BTV-3) was detected for the first time in cattle and sheep in southern England in 2023, the first UK BTV incursion for more than 15 years. Clinical signs were not observed, yet severe clinical disease and mortality were reported during recent BTV-3 outbreaks in northern Europe. METHODS:To investigate the clinical disease and infection kinetics associated with this UK BTV-3 strain, five British sheep were infected with a UK BTV-3 isolate using Culicoides biting midges. Clinical signs, pathology, infection dynamics, immune responses and Culicoides infection rates were assessed. RESULTS:All sheep were infected with BTV-3 and developed mild to moderate clinical bluetongue disease, characterised by fever, haemorrhagic diarrhoea, lameness, depression and widespread petechial haemorrhage. Three sheep reached clinical humane endpoints and were euthanased. Clinical signs/severity, infection kinetics and immune responses were highly variable. Infectious BTV-3 was isolated from sheep blood up to 28 days postinfection. LIMITATIONS:The impact of BTV-3 infection on British cattle and infection rate in UK Culicoides require investigation to fully determine the risk of this strain to UK livestock. CONCLUSIONS:This study confirms the potential impact of a BTV-3 incursion/outbreak on the UK sheep population, highlighting the need for an effective vaccine.

IntroductionBluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host.MethodsIn this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies.ResultsThe absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7.DiscussionThis study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

Since the reintroduction of African swine fever virus (ASFV) in Europe in 2007 and its subsequent spread to Asia, wild boar has played a crucial role in maintaining and disseminating the virus. There are significant gaps in the knowledge regarding infection dynamics and disease pathogenesis in domestic pigs and wild boar, particularly at the early infection stage. We aimed to compare domestic pigs and wild boar infected intranasally to mimic natural infection with one of the original highly virulent genotype II ASFV isolates (Armenia 2007). The study involved euthanising three domestic pigs and three wild boar on days 1, 2, 3, and 5 post-infection, while four domestic pigs and four wild boar were monitored until they reached a humane endpoint. The parameters assessed included clinical signs, macroscopic lesions, viremia levels, tissue viral load, and virus shedding in nasal and rectal swabs from day 1 post-infection. Compared with domestic pigs, wild boar were more susceptible to ASFV, with a shorter incubation period and earlier onset of clinical signs. While wild boar reached a humane endpoint earlier than domestic pigs did, the macroscopic lesions were comparatively less severe. In addition, wild boar had earlier viremia, and the virus was also detected earlier in tissues. The medial retropharyngeal lymph nodes were identified as key portals for ASFV infection in both subspecies. No viral genome was detected in nasal or rectal swabs until shortly before reaching the humane endpoint in both domestic pigs and wild boar, suggesting limited virus shedding in acute infections.

African swine fever (ASF) is an economically important disease due to high morbidity and mortality rates and the ability to affect all ages and breeds of pigs. Biosecurity measures to prevent the spread of the causative agent, African swine fever virus (ASFV), include prescriptive cleaning and disinfection procedures. The aim of this study was to establish the biocidal effects of twenty-four commercially available disinfectants including oxidizing agents, acids, aldehydes, formic acids, phenol, and mixed-class chemistries against ASFV. The products were prepared according to the manufacturer's instructions and a suspension assay was performed with ASFV strain, BA71V using Vero cells (African green monkey cells) to test efficacy in reducing ASFV infection of cells. Generally, disinfectants containing formic acid and phenolic compounds, as well as oxidizing agents reduced viral titers of ASFV by over 4 log10 at temperatures ranging from 4 °C to 20 °C. Hydrogen peroxide, aldehyde, and quaternary ammonium compounds containing disinfectants were cytotoxic, limiting the detection of viral infectivity reductions to less than 4 log10. These preliminary results can be used to target research on disinfectants which contain active ingredients with known efficacy against ASFV under conditions recommended for the country where their use will be applied.

A novel liquid stabiliser was tested with the Nigeria 75/1 Peste des Petit Ruminants (PPR) vaccine over two field studies carried out in sheep and goats. PPR seronegative sheep and goats were selected from farms surrounding Amman, Jordan and were vaccinated with either a stabilised liquid PPR vaccine that had been formulated 3 months prior to use and stored at 2-8 degrees C or a reconstituted lyophilised PPRV vaccine reconstituted on the day of vaccination. Sera were taken immediately before vaccination and at approximately 1.5, 3 and 6 months following vaccination, then subsequently tested using IDVet ID Screen & REG; PPR competition ELISA and Serum Neutralisation tests to determine the presence of PPRV anti-N antibodies and neutralising antibodies, respectively. It was observed that the liquid-stabilised vaccine was able to provide comparable antibody responses in both species to those induced by the lyophilized vaccine. The ability to store liquid stabilised PPRV vaccine for field use would positively impact PPRV eradication efforts.

In this study, we describe the epidemiological investigation of the first African swine fever (ASF) outbreak in a local domestic pig farm in the New Territories of Hong Kong in 2021. In the outbreak farm, several affected pigs presented clinical and pathological signs consistent with ASF, while the remaining pigs showed nonspecific clinical signs or did not exhibit any clinical signs. The relative low morbidity and mortality of ASF on this farm resulted in delayed detection and implementation of the control response. Despite this delay, no further spread of the disease from this farm to other farms or wild boars was observed. The clinical presentation of ASF in terms of morbidity and mortality on this farm indicated that it is essential for effective surveillance aimed at early detection for farmers, veterinarians, and pathologists to be educated about the different ways ASF can express itself in domestic pig populations. Epidemiological investigations consisted of field inspection, interviews with farm personnel to assess the management and biosecurity practices within the farm, and laboratory testing of animal and environmental samples. In addition, the complete genome of ASFV was obtained directly from the tissues of an infected pig to facilitate the epidemiological investigation. The genetic relationship at the whole genome level indicated that the isolate shared the highest level of similarity with genotype II ASFVs, including a 2019 isolate from Guangdong province, China (GD2019). Overall, the information presented here from the on-farm investigation with that from diagnostic testing and molecular analyses provides a basis for informed actions to prevent future incidents in farms with similar characteristics. Furthermore, this study highlighted the need to increase current knowledge about the molecular diversity amongst circulating viruses and potentially trace the source of infection.