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Background: Lumpy skin disease virus ( Poxviridae family- Capripoxvirus genus) is the aetiological agent of LSD, a disease primarily transmitted by hematophagous biting, affecting principally cattle. Currently, only live attenuated vaccines are commercially available, but their use is limited to endemic areas. There is a need for safer vaccines, especially in LSD-free countries. This research aims to develop and test a safe and efficacious inactivated vaccine. Moreover, in this study, we used keyhole limpet hemocyanin (KLH) as a positive marker to distinguish infected from vaccinated animals (DIVA). Methods: Lumpy skin disease virus was propagated on primary lamb testis cells and Madin-Darby bovine kidney cells (PLT and MDBK, respectively), and four inactivated vaccines were produced. The vaccines differed from each other with the addition or not of KLH and in cells used for virus propagation. To evaluate the safety and immunogenicity, the vaccines and two placebos were administered to six groups comprising six male calves each, and antibody response was investigated using both an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test. In addition, the LSD/γ-interferon test and KLH (IgM-IgG) ELISA were performed on the collected samples. Furthermore, the use of KLH allowed us to distinguish vaccinated animals in the ELISA results, without any interference on the strength of the immune response against the LSDV. Finally, the efficacy of one of four vaccines was investigated through a challenge, in which one group of vaccinated animals and one animal control group were infected with a live field strain of LSDV. Results: Four out of the six control animals showed severe clinical signs suggestive of LSD, and, therefore, were euthanized for overcoming the predetermined limit of clinical score. By contrast, the vaccinated animals showed only mild symptoms, suggesting a reduction in severe disease notwithstanding the incapability of the vaccine in reducing the virus shedding. Conclusion: The vaccines produced were safe and able to elicit both a humoral and a cellular immune response, characteristics that, together with the demonstrated efficacy, make our vaccine a good candidate for countering the LSD spread in disease-free countries, thus also facilitating disease containment throughout the application of a DIVA strategy.

Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4-8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge. The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals. This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the IZSAM., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013 and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to select the key viral and host processes associated with disease pathogenesis. We identified and experimentally validated five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, we showed that an agnostic machine learning approach can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Herder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lumpy skin disease (LSD) is a viral disease of cattle and water buffalo characterized by cutaneous nodules, biphasic fever, and lymphadenitis. LSD is endemic in Africa and the Middle East but has spread to different Asian countries in recent years. The disease is well characterized in cattle while little is known about the disease in buffaloes in which no experimental studies have been conducted. Six buffaloes and two cattle were inoculated with an Albanian LSD virus (LSDV) field strain and clinically monitored for 42 days. Only two buffaloes showed fever, skin nodules, and lymphadenitis. All samples collected (blood, swabs, biopsies, and organs) were tested in real-time PCR and were negative. Between day 39 and day 42 after inoculation, anti-LSDV antibodies were detected in three buffaloes by ELISA, but all sera were negative by virus neutralization test (VNT). Cattle showed severe clinical signs, viremia, virus shedding proven by positive real-time PCR results, and seroconversion confirmed by both ELISA and VNT. Clinical findings suggest that susceptibility in buffaloes is limited compared to in cattle once experimentally infected with LSDV. Virological results support the hypothesis of buffalo resistance to LSD and its role as an accidental non-adapted host. This study highlights that the sensitivity of ELISA and VNT may differ between animal species and further studies are needed to investigate the epidemiological role of water buffalo.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum . This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

The Istituti Zooprofilattici Sperimentali (IZSs) are public health institutes dealing with the aetiology and pathogenesis of infectious diseases of domestic and wild animals. During Coronavirus Disease 2019 epidemic, the Italian Ministry of Health appointed the IZSs to carry out diagnostic tests for the detection of SARS-CoV-2 in human samples. In particular, the IZS of Abruzzo and Molise (IZS-Teramo) was involved in the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by Real Time RT-PCR. Activities and infrastructures were reorganised to the new priorities, in a "One Health" framework, based on interdisciplinary, laboratory promptness, accreditation of the test for the detection of the RNA of SARS-CoV-2 in human samples, and management of confidentiality of sensitive data. The laboratory information system - SILAB - was implemented with a One Health module for managing data of human origin, with tools for the automatic registration of information improving the quality of the data. Moreover, the "National Reference Centre for Whole Genome Sequencing of microbial pathogens - database and bioinformatics analysis" - GENPAT - formally established at the IZS-Teramo, developed bioinformatics workflows and IT dashboard with ad hoc surveillance tools to support the metagenomics-based SARS-CoV-2 surveillance, providing molecular sequencing analysis to quickly intercept the variants circulating in the area. This manuscript describes the One Health system developed by adapting and integrating both SILAB and GENPAT tools for supporting surveillance during COVID-19 epidemic in the Abruzzo region, southern Italy. The developed dashboard permits the health authorities to observe the SARS-CoV-2 spread in the region, and by combining spatio-temporal information with metagenomics provides early evidence for the identification of emerging space-time clusters of variants at the municipality level. The implementation of the One Health module was designed to be easily modelled and adapted for the management of other diseases and future hypothetical events of pandemic nature., Competing Interests: The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (© 2022 Published by Elsevier B.V.)

In this report, we describe eight complete genome sequences of African horse sickness virus (AHSV) strains belonging to four different serotypes, namely, AHSV-5, AHSV-6, AHSV-8, and AHSV-9. Samples were collected in Namibia and South Africa from infected horses between 2000 and 2011. As expected, phylogenetic analyses of the variable outer capsid protein VP2 genomic sequences of AHSV-6 and AHSV-8 show higher nucleotide identity between the isolated viruses than that of the relevant reference strains. The full-genome sequence of AHSV will provide useful information on its geographical origin, and it will also be instrumental for comparing the distribution of the Namibian isolate with that of global isolates.

Monoclonal antibodies (MAbs) against epizootic hemorrhagic disease virus (EHDV) were produced by immunizing BALB/c mice with rec-VP7-EHDV2; 66 clones producing MAbs able to recognize the VP7-EHDV with a strong reaction were obtained and tested in indirect enzyme-linked immunosorbent assay (i-ELISA) against the whole epizootic hemorrhagic disease (EHD) virus serotype 2; potential cross-reactions with related orbiviruses, as Bluetongue virus (BTV) and African horse sickness virus (AHSV), were investigated as well by i-ELISA, Western blot, and immunofluorescence. Fifty-three MAbs were specific for EHDV (VP7 recombinant protein and whole virus) and 13 reacted also with the VP7 of BTV. None of the MAbs reacted with AHSV. MAbs specific for EHDV were further characterized in a competitive ELISA (c-ELISA): 20 among them were found useful to develop a c-ELISA for the detection of antibodies against EHDV in bovine sera. The availability of this extensive set of MAbs provides the opportunity to develop a c-ELISA for the serological diagnosis of EHDV and to tune new methods for the isolation and identification of the virus in biological samples and cell cultures. The experimentation protocol was approved by the Italian Ministry of Health (number 639/2018-PR, Resp. to Prot. BDF16.13#295833199#).

Rift Valley fever (RVF) is an emerging transboundary, mosquito-borne, zoonotic viral disease caused by a single serotype of a virus belonging to the Phenuiviridae family (genus Phlebovirus). It is considered an important threat to both agriculture and public health in endemic areas, because the virus, transmitted by different mosquito genera, leads to abortions in susceptible animal hosts especially sheep, goat, cattle, and buffaloes, resulting in severe economic losses. Humans can also acquire the infection, and the major sources are represented by the direct contact with infected animal blood, aerosol, consumption of unpasteurized contaminated milk and the bite of infected mosquitoes. Actually, the EU territory does not seem to be exposed to an imminent risk of RVFV introduction, however, the recent outbreaks in a French overseas department and some cases detected in Turkey, Tunisia and Libya, raised the attention of the EU for a possible risk of introduction of infected vectors. Thus, there is an urgent need to develop new therapeutic and/or preventive drugs, such as vaccines. In our work, we studied the immunogenicity of an inactivated and adjuvanted vaccine produced using a Namibian field strain of RVF virus (RVFV). The vaccine object of this study was formulated with Montanide Pet Gel A, a polymer-based adjuvant that has been previously reported for its promising safety profile and for the capacity to elicit a strong immune response. The produced inactivated vaccine was tested on six sheep and the level of IgM and IgG after the immunization of animals was evaluated by a commercial competitive ELISA, in order to assess the immunogenicity profile of our vaccine and to evaluate its potential use, as an alternative to the attenuated vaccines commercially available, in case of Rift Valley fever epidemic disease on EU territory. Following the administration of the second dose, 35 days after the first one, all animals seroconverted., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Anna Serroni et al.)