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The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

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.

The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of Cq values used to infer the presence of viral RNA in samples. This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR. When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.

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.

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.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015–2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0–88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4–62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

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.

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.