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Foot and Mouth Disease (FMD) is a viral disease, widespread and highly contagious affecting mainly cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to reduction in animals’ production such as drop of milk production, loss of body weight and high mortality rate in young ruminants. Sixteen nasal swabs and epithelial tissues were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Blood samples, swabs and epithelial tissues impressions on Flinders Technology Associates (FTA) cards were shipped to the FMD Reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards and molecular testing of the examined FTA cards confirmed that the FMD virus circulating in Libya was serotype O. Sequencing analysis of the FMD virus VP1 gene confirmed that FMD virus strain was serotype O/East Africa 3 (O/EA-3) topotype. The phylogenetic of the VP-1 gene of the FMD virus showed very high nucleotide identity (99.8%) between the virus circulating in Libya and the Algerian FMD virus strains isolated in Algeria on 2018 and 2019.

Foot-and-mouth disease (FMD) affects the livestock industry and socioeconomic sustainability of many African countries. The success of FMD control programs in Africa depends largely on understanding the dynamics of FMD virus (FMDV) spread. In light of the recent outbreaks of FMD that affected the North-Western African countries in 2018 and 2019, we investigated the evolutionary phylodynamics of the causative serotype O viral strains all belonging to the East-Africa 3 topotype (O/EA-3). We analyzed a total of 489 sequences encoding the FMDV VP1 genome region generated from samples collected from 25 African and Western Asian countries between 1974 and 2019. Using Bayesian evolutionary models on genomic and epidemiological data, we inferred the routes of introduction and migration of the FMDV O/EA-3 topotype at the inter-regional scale. We inferred a mean substitution rate of 6.64 × 10

Lumpy skin disease is a high-consequence disease in cattle caused by infection with the poxvirus lumpy skin disease virus (LSDV). The virus is endemic in most countries in Africa and an emerging threat to cattle populations in Europe and Asia. As LSDV spreads into new regions, it is important that signs of disease are recognized promptly by animal caregivers. This study describes the gross, microscopic, and ultrastructural changes that occur over time in cattle experimentally challenged with LSDV. Four calves were inoculated with wildtype LSDV and monitored for 19 to 21 days. At 7 days after inoculation, 2 of the 4 cattle developed multifocal cutaneous nodules characteristic of LSD. Some lesions displayed a targetoid appearance. Histologically, intercellular and intracellular edema was present in the epidermis of some nodules. Occasional intracytoplasmic inclusion bodies were identified in keratinocytes. More severe and consistent changes were present in the dermis, with marked histiocytic inflammation and necrotizing fibrinoid vasculitis of dermal vessels, particularly the deep dermal plexus. Chronic lesions consisted of full-thickness necrosis of the dermis and epidermis. Lesions in other body organs were not a major feature of LSD in this study, highlighting the strong cutaneous tropism of this virus. Immunohistochemistry and electron microscopy identified LSDV-infected histiocytes and fibroblasts in the skin nodules of affected cattle. This study highlights the noteworthy lesions of LSDV and how they develop over time.

Foot-and-mouth disease virus (FMDV) vaccines protect animals from infection by inducing antibodies. The level of neutralising antibody induced in response to vaccination (or infection), as measured by a virus neutralisation test, is an important parameter with regards to the level of protection afforded against subsequent challenge. However, in addition to overall titre, antibody avidity also represents a crucial metric when assessing the protective efficacy of antibodies. In this project we investigated the use of biolayer interferometry (BLI) to measure the avidity of FMDV antibodies to FMDV antigens. Antibodies targeting site I of the FMDV particle were detected using a commercially synthesised biotinylated peptide. In contrast, the entire antigenic landscape of the FMDV particle was represented by biotinylated FMDV capsids. The antigens were loaded onto Octet streptavidin biosensors at an optimal concentration prior to dipping into antibodies. The sera from different animals varied in avidity, reflecting the quantitative differences in avidity that exist between individual animals in response to FMDV vaccines. Interestingly, the Kdis values obtained for site I vs the entire capsid were different, supporting the importance of other sites beyond site I. Similarly, monoclonal antibodies targeting distinct, known antigenic sites on the capsid surface also resulted in different avidities. The BLI methodology reported here offers a useful approach by which to investigate the strength of antibody interactions at specific sites. In conjunction with recombinant technology, BLI will help aid in investigations into the relative importance of the different antigenic sites with regards to inducing a protective response.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012–2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVβ6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

Advances in the epidemiological tracing of pathogen transmission have been largely driven by the increasing characterisation of whole-genome sequence data obtained at a finer resolution from infectious disease outbreaks. Dynamic models that integrate genomic and epidemiological data further enhance inference on the evolutionary history and transmission dynamics of epidemic outbreaks by reconstructing the network of ‘who-infected-whom’. Swine Vesicular Disease (SVD) was present in Italy from 1966 until 2015, and since the mid-1990s, it has mainly been circulating within Italy’s central-southern regions with sporadic incursions to the north of the country. However, a recrudescence of SVD in northern Italy was recorded between November 2006 and October 2007, leading to a large-scale epidemic that significantly affected the intensive pig industry of the Lombardy region. In this study, by using whole-genome sequence data in combination with epidemiological information on disease occurrences, we report a retrospective epidemiological investigation of the 2006–2007 SVD epidemic, providing new insights into the transmission dynamics and evolutionary mode of the two phases that characterised the epidemic event. Our analyses support evidence of undetected premises likely missed in the chain of observed infections, of which the role as the link between the two phases is reinforced by the tempo of SVD virus evolution. These silent transmissions, likely resulting from the gradual loss of a clear SVD clinical manifestation linked to sub-clinical infections, may pose a risk of failure in the early detection of new cases. This study emphasises the power of joint inference schemes based on genomic and epidemiological data integration to inform the transmission dynamics of disease epidemics, ultimately aimed at better disease control.

Foot-and-mouth disease (FMD) is a highly contagious animal disease caused by an RNA virus subdivided into seven serotypes that are unevenly distributed in Asia, Africa, and South America. Despite the challenges of controlling FMD, since 1996 there have been only two outbreaks attributed to serotype C, in Brazil and in Kenya, in 2004. This article describes the historical distribution and origins of serotype C and its disappearance. The serotype was first described in Europe in the 1920s, where it mainly affected pigs and cattle but as a less common cause of outbreaks than serotypes O and A. No serotype C outbreaks have been reported in Europe since vaccination stopped in 1990. FMD virus is presumed to have been introduced into South America from Europe in the nineteenth century, although whether serotype C evolved there or in Europe is not known. As in Europe, this serotype was less widely distributed and caused fewer outbreaks than serotypes O and A. Since 1994, serotype C had not been reported from South America until four small outbreaks were detected in the Amazon region in 2004. Elsewhere, serotype C was introduced to Asia, in the 1950s to the 1970s, persisting and evolving for several decades in the Indian subcontinent and for eighteen years in the Philippines. Serotype C virus also circulated in East Africa between 1957 and 2004. Many serotype C viruses from European and Kenyan outbreaks were closely related to vaccine strains, including the most recently recovered Kenyan isolate from 2004. International surveillance has not confirmed any serotype C cases, worldwide, for over 15 years, despite more than 2,000 clinical submissions per year to reference laboratories. Serology provides limited evidence for absence of this serotype, as unequivocal interpretation is hampered by incomplete intra-serotype specificity of immunoassays and the continued use of this serotype in vaccines. It is recommended to continue strengthening surveillance in regions of FMD endemicity, to stop vaccination against serotype C and to reduce working with the virus in laboratories, since inadvertent escape of virus during such activities is now the biggest risk for its reappearance in the field.

We conducted a serologic survey among dogs and cats in Italy to detect antibodies against severe acute respiratory syndrome virus 2 (SARS-CoV-2). We found that SARS-CoV-2 seroprevalence was higher among cats (16.2%) than dogs (2.3%). In addition, seroprevalence was higher among animals living in close contact with SARS-CoV-2-positive owners.

The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.

Swine vesicular disease (SVD) is a contagious viral disease of pigs clinically indistinguishable from other vesicular diseases, such as foot and mouth disease, vesicular stomatitis, vesicular exanthema of swine, and idiopathic vesicular disease. In Italy, where SVD was first reported in 1966, an eradication program started in 1995. The program, updated in 2008, was based on regionalization, complete control on pig movements, improvement of pig farms biosecurity, appropriate cleansing and disinfection procedures of vehicles approved for pig transportation, and a testing program using both serological and virological assays. In cases of confirmed SVD virus infection a stamping-out policy was applied. In the period 2009 to 2019, between 300,000 and 400,000 pigs were serologically tested each year. The last SVD outbreak was notified in 2015, and the last seropositive pig was detected in 2017. SVD surveillance is still ongoing and no proof of virus activity has been detected so far. All available data support the complete SVD virus eradication from the Italian pig industry.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents., Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.

This study describes the development and validation of a simplified enzyme-linked immunosorbent assay (ELISA) for the detection and discrimination of foot-and-mouth disease virus (FMDV) serotypes O, A, C and Asia 1. The multiplex ELISA was designed using selected, type-specific monoclonal antibodies (MAbs) coated onto ELISA plates as catching antibodies and a unique pan-FMDV MAb (1F10) as detector conjugate. Capture MAbs with the broadest intratypic reactivity were selected for each of the four FMDV serotypes by screening large panels of candidate MAbs with a wide spectrum of representative FMDV isolates. An additional pan-FMDV ELISA using 1F10 MAb for both capture and detection was used to complement the specific typing ELISAs to detect virus isolates, which might escape binding to the selected serotype-specific MAbs. This multiplex ELISA was prepared in a stabilized format, with immunoplates pre-coated with six MAbs and positive antigen controls already trapped by the relevant MAb, with the view to make available a diagnostic kit. Diagnostic performance of the MAbs-multiplex ELISA was analysed using 299 FMDV-positive epithelial suspensions representative of the antigenic and genomic variability within each serotype. Overall, the results provided evidence that the diagnostic performance of this assay platform is improved compared to the more complex polyclonal-based antigen detection ELISA; combining data from different serotypes and referring to the gold standard tests (i.e. virus isolation and/or RT-PCR), the MAbs-multiplex ELISA showed a sensitivity of 79% for the serotype-specific ELISA, compared to 72% for the polyclonal ELISA. The absence of reactivity of a minority of FMDV strains using the MAbs-multiplex ELISA can largely be attributed to deteriorated or low antigen concentration in the sample. This multiplex ELISA is simple, rapid and stable. FMDV antigenic diversity was adequately covered by the selected MAbs. Therefore, it can be used to replace existing polyclonal ELISAs for FMDV detection and serotyping.

Porcine epidemic diarrhoea virus (PEDV), belongs to the genus Alphacoronavirus in the family Coronaviridae and causes severe diarrhoea, vomiting, dehydration and high mortality in seronegative newborn piglets. Thus, a precise and rapid diagnosis of PEDV infection is important for the application of control measures to limit viral dissemination. In this investigation, a monoclonal antibodies (MAbs)-based competitive enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against PEDV was developed and validated. The diagnostic performance of the test was evaluated by receiver operating characteristic (ROC) analysis using a panel of 829 known sera collected from different commercial pig farms, with or without a history of PED presence and from experimentally infected pigs. The competitive ELISA showed excellent diagnostic performance and discriminatory power with high sensitivity (Se) and specificity (Sp) values (Se = 96.5%, 95% IC 94.1–98.1; Sp = 98.2%, 95% IC 96.3–99.3). Moreover, this competitive ELISA method has other properties, such as high feasibility of testing sera without pre-treatment and automatic and instrument-mediated revealing, that makes it appropriate for large-scale screenings of affected pig farms in endemic regions or for monitoring plans in PEDV-free areas., Highlights • A monoclonal antibodies (MAbs)-based competitive ELISA for detecting antibodies against PEDV was developed and validated. • The diagnostic performance of the test was evaluated by ROC analysis using a panel of 829 known sera. • The ELISA showed excellent diagnostic performance and discriminatory power.

This study describes the genetic characterization of serotype A viruses collected during outbreaks of foot-and-mouth disease (FMD) that occurred in Algeria in 2017. These are the first reports of clinical cases due to this serotype in the country since 1977. One complete genomic sequence (comprising 8,119 nucleotides) and three additional near-complete genomic sequences were generated. Phylogenetic analyses demonstrated that these viruses were classified within the A/AFRICA/G-IV lineage, most closely related to viruses circulating in Nigeria between 2009 and 2015. These unexpected results motivate further studies to define the precise pathways by which this viral lineage has been introduced into North Africa in order to understand risks of future disease incursions into the region.

Outbreaks of foot-and-mouth disease (FMD) in North Africa (2013) and the Gulf States (2013) of the Middle East have been caused by a FMD viral lineage (O/ME-SA/Ind-2001) that was before 2013 restricted to the Indian Sub-continent. This study was undertaken to assess the in vivo efficacy of a FMD virus emergency vaccine type O1 Manisa against heterologous challenge with a representative field virus (O/ALG/3/2014) from this emerging lineage. This widely available vaccine was selected since in vitro vaccine-matching results gave inconclusive results as to whether or not it would be protective. Three groups of five cattle were vaccinated with O1 Manisa (homologous potency ≥6PD50/dose) using study guidelines outlined in the European Pharmacopeia, and challenged at 21 days post-vaccination by tongue inoculation. All animals that were vaccinated with the lowest dose (1/16) of vaccine developed generalised FMD, defined as vesicular lesions at the feet. One animal vaccinated with a 1/4 dose of the vaccine also developed generalised disease, as did two animals vaccinated with the full dose of vaccine. These results indicate that the heterologous potency of this high potency O1 Manisa vaccine was approximately 3.5 PD50/dose. These data support the use of the O1 Manisa vaccine for FMD control in areas where FMDV is endemic e.g. North Africa, and motivate further studies to evaluate other vaccine candidates (or multivalent combinations) that might be potentially used for emergency purposes in FMD-free settings.

International audience; Serologic assays used to detect antibodies to nonstructural proteins (NSPs) of foot-and-mouth disease virus (FMDV) are used for disease surveillance in endemic countries, and are essential to providing evidence for freedom of the disease with or without vaccination and to recover the free status of a country after an outbreak. In a 5-site inter-laboratory study, we compared the performance of 2 commercial NSP ELISA kits (ID Screen FMD NSP ELISA single day [short] and overnight protocols, ID.Vet; PrioCHECK FMDV NS antibody ELISA, Thermo Fisher Scientific). The overall concordance between the PrioCHECK and ID Screen test was 93.8% (95% CI: 92.0–95.2%) and 94.8% (95% CI: 93.1–96.1%) for the overnight and short ID Screen incubation protocols, respectively. Our results indicate that the assays (including the 2 different formats of the ID Screen test) can be used interchangeably in post-outbreak serosurveillance.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral non-structural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus serotype and their sensitivity performances may be affected by antigenic variability within each serotype and mismatching between tests reagents. As a consequence, FMD Reference Laboratories need to maintain contingency to employ multiple type-specific assays for large-scale serological surveillance and post-vaccination monitoring in the event of FMD outbreaks. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterised using a panel of intertypic-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide ELISA (VP2-ELISA) was optimised using experimental and reference antisera from immunized, convalescent and negative animals (n=172). The VP2-ELISA is universal, simple and provided sensitive (98.6 %) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for sero-surveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in endemic countries.

An increase in autochthonous hepatitis E virus (HEV) infections has been recorded in Italy suspected to be zoonotically transmitted from pigs; this study was carried out to determinate the seroprevalence and risk factors associated with hepatitis HEV exposition, both in swine and humans working in pig farms, located within a high-density pig farming area in Piedmont region, north-western Italy. The presence of viral RNA in human and swine samples was also evaluated, and phylogenetic analysis was performed on HEV-positive samples. Forty-two swine farms were sampled; 142 workers were enrolled in the study and classified into two groups: (i) 69 workers with occupational contact with swine (including veterinarians and farmers) recruited in the 42 sampled farms; (ii) 73 without occupational contact with swine. Forty-one of 42 (97%) swine farms resulted positive to enzyme-linked immunosorbent assay test for HEV antibodies (Abs). Overall seroprevalence in swine was 50% (441/879), with seropositivity rate higher in sows (333/469, 71%). HEV RNA in stool samples was detected in animals from 13 of 42 tested farms (31%), and a higher positivity resulted in weaners (40/246, 16.3%). Phylogenetic analysis classified all HEV isolates within genotype 3 (subtypes 3f, 3e, 3c). All humans were negative for HEV viral genome in blood. Five of 142 sera were positive for IgG anti-HEV with an overall prevalence of 3.52% with no statistically significant differences in prevalence rates between workers at zoonotic risk and the control group (5.7% versus 1.3%). In contrast, a significant difference (OR 10.1) was observed within the subgroup including subjects exposed for short periods (veterinarians) compared with those who worked for long periods (farmers) suggesting a correlation between the time of exposure and the likelihood of HEV infection. Reporting HEV infection is not mandatory in Italy, but a constant epidemiological surveillance should be ensured to clarify the epidemiology of this disease.

Orthobunyaviruses are enveloped viruses that are arthropod-transmitted and cause disease in humans and livestock. Viral attachment and entry are mediated by the envelope glycoproteins Gn and Gc, and the major glycoprotein, Gc, of certain orthobunyaviruses is targeted by neutralizing antibodies. The domains in which the epitopes of such antibodies are located on the glycoproteins of the animal orthobunyavirus Schmallenberg virus (SBV) have not been identified. Here, we analysed the reactivity of a set of mAbs and antisera against recombinant SBV glycoproteins. The M-segment-encoded proteins Gn and Gc of SBV were expressed as full-length proteins, and Gc was also produced as two truncated forms, which consisted of its amino-terminal third and carboxyl-terminal two-thirds. The sera from convalescent animals reacted only against the full-length Gc and its subdomains and not against the SBV glycoprotein Gn. Interestingly, the amino-terminal domain of SBV-Gc was targeted not only by polyclonal sera but also by the majority of murine mAbs with a neutralizing activity. Furthermore, the newly defined amino-terminal domain of about 230 aa of the SBV Gc protein could be affinity-purified and further characterized. This major neutralizing domain might be relevant for the development of prophylactic, diagnostic and therapeutic approaches for SBV and other orthobunyaviruses.

A cross-sectional study was carried out to determine foot-and-mouth disease (FMD) seroprevalence and identify risk factors of exposure among cattle herds raised in three zones with different types of land use and progressively distant from the Maasai Mara National Reserve (MMNR) boundary. We selected five villages purposively; two in zone 1 (area20 km from the MMNR), another two in zone 2 (area between 20-40 km away from the MMNR) and one in zone 3 (area40 km away from the MMNR). A total of 1170 cattle sera were collected from 390 herds in all the zones and tested for antibodies against the non-structural proteins (NSPs) of FMD virus (FMDV) using two 3ABC-based Enzyme-Linked Immunosorbent Assay ELISA kits. All sera samples were also screened for serotype-specific antibodies using Solid Phase Competitive ELISA (SPCE) kits (IZSLER, Italy). We targeted FMDV serotypes A, O, South African Territory [SAT] 1 and SAT 2, known to be endemic in East Africa including Kenya. Data on putative risk factors for FMD seropositivity in cattle were collected using a questionnaire. The overall apparent animal-level FMD seroprevalence based on the parallel comparison of the two anti-NSPs ELISA kits was 83.8 % (95 % CI; 81.8-85.9), and differed significantly across zones. Zone 1 had a higher seroprevalence than zones 2 and 3 (χ

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb-based competitive ELISA for the detection of antibodies against IDV. Thirty-one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724-3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid-phase competitive ELISA (IDV-cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase-conjugated MAb as competitor, which had shown wide intratype cross-reactivity and positivity in HI. To evaluate the diagnostic performances of IDV-cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut-off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV-cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI -0.899 to 0.961) between HI test and IDV-cELISA. ROC analysis showed that IDV-cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993-1.000). A cut-off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1-99.9) and 98.75 (95% CI 97.1-99.6). These results proved excellent diagnostic performances of IDV-cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti-IDV antibodies in different animal species.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock affecting animal production and trade throughout Asia and Africa. Understanding FMD virus (FMDV) global movements and evolution can help to reconstruct the disease spread between endemic regions and predict the risks of incursion into FMD-free countries. Global expansion of a single FMDV lineage is rare but can result in severe economic consequences. Using extensive sequence data we have reconstructed the global space-time transmission history of the O/ME-SA/Ind-2001 lineage (which normally circulates in the Indian sub-continent) providing evidence of at least 15 independent escapes during 2013–2017 that have led to outbreaks in North Africa, the Middle East, Southeast Asia, the Far East and the FMD-free islands of Mauritius. We demonstrated that sequence heterogeneity of this emerging FMDV lineage is accommodated within two co-evolving divergent sublineages and that recombination by exchange of capsid-coding sequences can impact upon the reconstructed evolutionary histories. Thus, we recommend that only sequences encoding the outer capsid proteins should be used for broad-scale phylogeographical reconstruction. These data emphasise the importance of the Indian subcontinent as a source of FMDV that can spread across large distances and illustrates the impact of FMDV genome recombination on FMDV molecular epidemiology.