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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 (area < 20 km from the MMNR), another two in zone 2 (area between 20-40 km away from the MMNR) and one in zone 3 (area >40 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 2  = 116.1, df = 2, p < 0.001). In decreasing order, the overall seroprevalences of FMDV serotypes A, SAT 2, O and SAT 1 were 26.3 % (95 % CI; 23.5-29.2), 21.4 % (95 % CI; 18.8-24.0), 21.2 % (95 % CI; 18.7-23.9) and 13.1 % (95 % CI; 11.1-15.3), respectively. The distribution of these serotypes differed significantly between zones (p < 0.05) except for SAT 2 serotype (χ 2  = 0.90, df = 2, p = 0.639). Both serotypes A and O were more prevalent in zones 1 and 2 than zone 3 while serotype SAT 1, was higher in zone 3 compared to other zones. The results of multivariable analyses identified animal sex (i.e., female), raising of cattle in zones 1 and 2 (areas < 40 km away from the MMNR); mixing of cattle from multiple herds at watering points, and pastoral husbandry practices, as significant predictors of animal-level FMD seropositivity. This study established that FMD seroprevalence declined with distance from the MMNR., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2019 Her Majesty the Queen in Right of Canada Transboundary and Emerging Diseases © 2019 Blackwell Verlag GmbH.)

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.

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., (© 2018 Blackwell Verlag GmbH.)

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., (© 2018 Blackwell Verlag GmbH.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

The effective control of foot-and-mouth disease (FMD) requires sensitive, specific and rapid diagnostic tools. However, the control and eradication of FMD in Africa is complicated by, among other factors, the existence of five of the seven FMD virus (FMDV) serotypes, including the SAT-serotypes 1, 2 and 3 that are genetically and antigenically the most variable FMDV serotypes. A key diagnostic assay to enable a country to re-gain its FMD-free status and for FMD surveillance, is the 3ABC or the non-structural protein (NSP) enzyme-linked immunosorbent assay (ELISA). Although many kits are available to detect 3ABC antibodies, none has been developed specifically for the variable SAT serotypes. This study designed a SAT-specific NSP ELISA and determined whether this assay could better detect NSP-specific antibodies from FMDV SAT-infected livestock. The assay's performance was compared to validated NSP assays (PrioCheck®-NSP and IZSLER-NSP), using panels of field and experimental sera, vaccinated and/or infected with FMDV SAT1, SAT2 or SAT3. The sensitivity () of the SAT-NSP was estimated as 76% (70%, 81%) whereas the specificity was 96% (95%, 98%) at a 95% confidence interval. The sensitivity and specificity were comparable to the commercial NSP assays, PrioCheck®-NSP (82% and 99%, respectively) and IZSLER-NSP (78% and 98%, respectively). Good correlations were observed for all three assays., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We conducted a cross-sectional study during 2013 to quantify the serological prevalence of peste des petits ruminants (PPR) infection and to investigate host factors associated with PPR infection in small ruminants in Libya. A two-stage sampling design was carried out. A total number of 148 flocks owning at least 100 heads each were randomly selected. Sixteen to forty-eight samples were collected from each selected flock. A total number of 3,508 serum samples from unvaccinated animals were collected and analysed at IZSLER Brescia, Italy, by using competitive ELISA, IDvet innovative diagnostics (IDvet 310, France). The overall serological prevalence among SR was 33% (95% CI: 31.4-34.5). Significant differences between the prevalence in the geographical branches were observed. The lowest prevalence level was observed in Zawiyah branch (16.1%), whereas the highest value was obtained for the Sabha branch (56.8%). Considering the age, a serological prevalence of 24.7%, 31.5% and 42.1% was observed in SR <1 year, between 1 and 2 years and more than 2 years, respectively. Statistically significant differences (p < .001) in the sero-prevalence levels were also observed between the age groups. Our findings suggest that the southern part of Libya could be more exposed to the infections coming from the neighbouring countries and this should be better investigated to correctly identify wherever specific entry points can be considered at higher risk than others. The results also confirmed the endemic status of PPR in Libya, with a constant exposure to the infection of the animals during their life. In the framework of the global strategy for control and eradication of PPR, our results, even if obtained by a preliminary study, can contribute to the assessment of the epidemiological situation of PPR in Libya as required by the Stage 1 of the plan., (© 2017 Blackwell Verlag GmbH.)

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., (© 2016 Blackwell Verlag GmbH.)

Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus (SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S-S) were identified and the deletion of the third S-S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.

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 O 1 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 O 1 Manisa (homologous potency ≥6PD 50 /dose) using study guidelines outlined in the European Pharmacopeia, and challenged at 21days 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 O 1 Manisa vaccine was approximately 3.5 PD 50 /dose. These data support the use of the O 1 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sporadic outbreaks of foot-and-mouth disease (FMD) have occurred in Libya for almost fifty years. During the spring of 2013, a countrywide serosurvey was undertaken to assess the level of FMD virus circulation and identify FMD virus serotypes in the country. A total of 4221 sera were collected, comprising samples from large ruminants (LR; n=1428 samples from 357 farms) and small ruminants (SR; n=2793 samples from 141 farms). FMD sero-prevalence of NSP antibodies determined by ELISA were 19.0% (271/1428) with 95% CI (16.9 - 21.0) and 13.5% (378/2793) with 95% CI (12.3 - 14.8) for LR and SR samples, respectively. The sero-prevalence of NSP antibodies in LR was 12.3% and 19.8% for age group < 1 year and ≥ 1 year, respectively (X 2 = 4.95, P= 0.026), while in SR was 3.7%, 13.6% and 21.3% for age group < 1 year, 1-2 year and > 2 year, respectively (X 2 = 118.1, P= 0.000). These observed NSP serologic profiles support the hypothesis of an endemic level of FMD circulation in Libya. All positive sera were tested for SP antibodies for O, A and SAT-2 FMD virus serotypes. Serotype O was the dominant circulating serotype followed by serotype A, while evidence of SAT-2 was not found. These data provide an insight into the wider epidemiology of FMD in Libya, and contribute to field and laboratory investigations that during 2013 serotype O (O/ME-SA/Ind-2001 lineage) was isolated from clinical samples collected from the country.

The Simbu serogroup of orthobunyaviruses comprises a wide range of viruses with different medical and veterinary relevance. These viruses are known to reassort, and coinfection of the same cell is one of the prerequisites for reassortment. Here, a mammalian cell line was infected with various members of this virus group, inoculated after several time points with a second Simbu serogroup virus, and analyzed by strain or species specific immunofluorescence staining. Different virus species or different strains of the same virus species were able to co-infect mammalian cells, but only for a limited time frame. After a few hours, the replication of the first virus led to a gradual inhibition of a second virus until a complete resistance to superinfection after 24h regardless whether it is another strain of the same virus species or a distinct member of the serogroup., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia., (© 2014 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvβ6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvβ6 and FMDV empty capsids in FMD diagnostic assays.

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.

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics.

European brown hare syndrome virus (EBHSV) is the aetiological agent of European brown hare syndrome (EBHS), a disease affecting Lepus europaeus and Lepus timidus first diagnosed in Sweden in 1980. To characterize EBHSV evolution we studied hare samples collected in Sweden between 1982 and 2008. Our molecular clock dating is compatible with EBHSV emergence in the 1970s. Phylogenetic analysis revealed two lineages: Group A persisted until 1989 when it apparently suffered extinction; Group B emerged in the mid-1980s and contains the most recent strains. Antigenic differences exist between groups, with loss of reactivity of some MAbs over time, which are associated with amino acid substitutions in recognized epitopes. A role for immune selection is also supported by the presence of positively selected codons in exposed regions of the capsid. Hence, EBHSV evolution is characterized by replacement of Group A by Group B viruses, suggesting that the latter possess a selective advantage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hepatitis E virus (HEV) is a RNA non-enveloped virus that comprises four genotypes. The genome of HEV is organized into three Open Reading Frames (ORFs), and the ORF2 is responsible for encoding capsid proteins. HEV can infect a wide range of hosts, and pigs are considered the main reservoir. HEV infection is considered a zoonosis and it is responsible for acute hepatitis in humans, especially in developing countries. The development of a blocking ELISA would be of high value for screening purpose, because there is no need of species specific reagents. The present study was conducted to assess three in-house ELISAs for the detection of HEV infection in 779 sera collected from breeding and fattening farms under field conditions. Two assays were indirect ELISAs, while the third was a blocking ELISA. Two different recombinant antigens were generated from specific sequences of the HEV-ORF2, and a Latent Class approach in a Bayesian framework was used to evaluate the diagnostic accuracy of each ELISA. Because the three ELISAs cannot be thought of as independent, all possible dependence structures were modelled starting from the general case of conditional independence to the most complex situation of three mutually dependent assays. Results showed that none of the three ELISAs was significantly superior to the others in terms of sensitivity (posterior median value ranging from 89% to 94%, all 95% posterior credible intervals (95%PCI) overlapped). In terms of specificity, one of the indirect ELISAs was superior to blocking ELISA (posterior median indirect ELISA: 99%, 95%PCI: 98-100%; blocking ELISA: 90%; 95%PCI: 86-94%). However, this difference could be due to the potential wider spectrum of antibodies that blocking ELISA can actually detect., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvβ6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle., Importance: The objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

The role of the GPI-anchor in prion disease pathogenesis is still a challenging issue. In vitro studies have shown that anchorless cellular prion protein (PrP(C)) undergoes aberrant post-translational processing and metabolism. Moreover, transgenic (Tg) mice overexpressing anchorless PrP(C) develop a spontaneous neurological disease accompanied with widespread brain PrP amyloid deposition, in the absence of spongiform changes. Generation of PrP forms lacking the GPI and PrP amyloidosis are striking features of human stop codon mutations in the PrP gene (PRNP), associated with PrP cerebral amyloid angiopathy (PrP-CAA) and Gerstmann-Sträussler-Scheinker (GSS) syndrome. More recently, the presence of anchorless PrP species has been also claimed in sporadic Creutzfeldt-Jakob disease (sCJD). Using a highly sensitive protein separation technique and taking advantage of reference maps of synthetic PrP peptides, we investigated brain tissues from scrapie-infected "anchorless PrP" Tg mice and wild type mice to determine the contribution of the GPI-anchor to the molecular mass and isoelectric point of PrP quasispecies under two-dimensional electrophoresis. We also assessed the conformational properties of anchorless and anchored prions under standard and inactivating conditions. These studies were extended to sCJD and GSS. At variance with GSS, characterization of PrP quasispecies in different sCJD subtypes ruled out the presence of anchorless prions. Moreover, under inactivating conditions, mice anchorless prions, but not sCJD prions, generated internal PrP fragments, cleaved at both N and C termini, similar to those found in PrP-CAA and GSS brain tissues. These findings show that anchorless PrP(Sc) generates GSS-like PrP fragments, and suggest a major role for unanchored PrP in amyloidogenesis.

Caprine herpesvirus 1 (CpHV1) is a member of ruminant alphaherpesviruses antigenically related to the prototype bovine herpesvirus 1 (BoHV1). Although cross reactivity between the two viruses involves many structural glycoproteins, the use of two competitive BoHV1 ELISAs detecting anti gB and gE antibodies has been proposed for CpHV1 infection, resulting mainly in a gB+/gE- reactivity and leading to suppose that CpHV1 gE may represent an useful target for the development of specific diagnostic test. Since CpHV1 gE gene has been only partially characterized so far, in this study the genome fragment of the short unique unit (Us) encompassing gI and gE gene was amplified and sequenced. Gene fragments encoding the ectodomain of both glycoproteins were subcloned into pSECTag2/Hygro and expressed in HEK293T cells as secreted form in serum free medium. Due to the lack of specific monoclonal antibodies (Mabs), the same recombinant glycoproteins were obtained from BoHV1 and used as positive control with a panel of specific gE and gI Mabs as well as in some ELISA assays. Results clearly indicate that the ectodomain of CpHV1 gE, immobilized on solid face in an indirect ELISA format, represents a sensitive and specific marker of infection, when compared with neutralization test, with absence of very low degree of cross-reactivity with BoHV1 gE counterpart, while the use of CpHV1 gI-ELISA or a combination of gE/gI complex did not significantly improve the sensitivity of the assay. In addition, in the rare event in which cross species barrier occurs for both viruses from their natural host to other species, the use of both BoHV1 and CpHV1 gE in a comparative assay may be proposed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Knowledge of the antigenic structure of foot-and-mouth disease virus (FMDV) has relevance in the development of diagnostic assays, in the evaluation of the antigenic variability and in the selection of appropriate vaccine strains. Antigenic sites have been investigated only in FMDVs of serotypes O, A and C, while it would be valuable to extend studies also to other serotypes. This paper reports the identification of antigenic sites involved in virus neutralization in the FMDV serotype Asia 1 by using a new panel of mAbs and their relation with sites described in other serotypes is discussed. Out of 24 mAbs raised against the FMDV serotype Asia 1, 10 neutralize viral infectivity and were used to select FMDV mutants resistant to neutralization. On the basis of their reactivity profile with virus mutants, the 10 neutralizing mAbs were clustered in four groups corresponding to four independent antigenic sites. By comparing the amino acid sequence of the parental virus and of virus mutants, the amino acids crucial for the four sites were mapped at the following positions: VP1 140-142, VP2 67-79, VP3 58/59 and VP3 218. Three of the four neutralizing sites identified and mapped on FMDV serotype Asia 1 correspond structurally and functionally to analogous sites described in FMDV serotypes O, A and C, enforcing the evidence that these are dominant antigenic sites in the FMDV structure. The fourth site, located at the C terminus of VP3, is a new independent site, described for the first time in FMDV.

Diagnostic and containment measures are essential for the management of avian influenza. In this study, a monoclonal antibody (MAb)-based competitive ELISA for detecting antibodies against H5 avian influenza viruses was developed and validated. Twenty-five anti-H5 MAbs were characterised using competitive, indirect and sandwich ELISAs, immunofluorescence, Western blotting and virus neutralisation and haemagglutination inhibition assays. One MAb (5D8) with wide intra-subtype cross-reactivity was selected and characterised using escape mutant selection. Epitope analysis showed that this MAb recognises a conformational epitope comprising amino acid residues in positions 170, 235 and 240 located in the receptor binding domain. The diagnostic performance of the test was evaluated by ROC analysis using a panel of 950 known sera collected from different avian species, including chickens, turkeys, ducks, pheasants, wild Anseriformes and ostriches. The competitive ELISA had excellent diagnostic performance and discriminatory power with high Se and Sp values (Se: 99.6-95% CI 98.0-100; Sp: 99.4-95% CI 98.5-99.8). In addition to its excellent diagnostic performance, properties of the competitive ELISA, such as high feasibility of testing sera without pre-treatment and potential for automation and instrument-mediated detection, make it ideal for screening samples, confirming positive HI assay results or analysing samples that are difficult to test using the HI assay., (Published by Elsevier B.V.)

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.

The gene encoding the VP2 protein of canine parvovirus type 2 was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination, Western blotting and hemagglutination inhibition test, using Canine parvovirus type-2 (CPV-2) positive sera. An enzyme-linked immunosorbent assay (ELISA) using the rVP2 was used for testing CPV-2 positive and negative sera from dogs and for determining the threshold of maternally derived antibodies interfering with successful vaccination of pups against CPV-2., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs., Methods: Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity., Results: All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs., Conclusions: MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

A sandwich ELISA using recombinant integrin αvβ6 as a capture ligand and serotype-specific monoclonal antibodies (Mabs) as detecting reagents has been compared with a polyclonal antibody based ELISA (using type-specific rabbit antibodies as capture and guinea pig antibodies as detectors), which is employed routinely at the FAO World Reference Laboratory for Foot-and-Mouth Disease (FMD), for the identification and serotyping of FMD virus (FMDV). The study used cell culture grown antigens (1351 FMDV positive) derived from suspected cases of vesicular disease collected from 86 countries between 1924 and 2011, those positive for the other vesicular diseases of swine vesicular disease (n = 25) and vesicular stomatitis (n = 45) and negative samples collected from uninfected cell cultures (n = 36). The diagnostic sensitivity of the assays was similar at 98.1% (polyclonal ELISA) compared to 97.9% (integrin/Mab ELISA) but the serotypic-specificity of the latter was vastly superior (96%) to that of the former (61.5%). Reactions with the viruses of swine vesicular disease and vesicular stomatitis, which produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The integrin/Mab ELISA recognized FMDV strains of wide antigenic and molecular diversity of all seven serotypes and although some FMDV isolates were not detected, the greater specificity of the assay, while retaining test sensitivity comparable to the conventional assay, warrants its consideration for adoption for routine diagnostic use., (Copyright © 2011 Elsevier B.V. All rights reserved.)

At the end of 2006, a recrudescence of swine vesicular disease (SVD) was recorded in Italy and the disease spread widely throughout the northern regions. Lombardy, a densely populated pig area, was most affected and the presence of the disease caused heavy economic losses to the entire pig industry. Although SVD is considered only moderately contagious, the epidemic in the north was characterised by a rapid spread of the condition. Numerous difficulties were encountered in eradicating it. Over the past decade, there has been a significant increase in the population of pigs in Lombardy, concentrated mainly in a few areas which were the most severely affected during the 2006 to 2007 SVD epidemic. Increases in both the pig population and animal movements, combined with weak biosecurity measures, increased the spread rate of the disease and hampered eradication activities.

Unsedated colonoscopy is available worldwide, but is not a routine option in the United States (US). We conducted a literature review supplemented by our experience and expert commentaries to provide data to support the use of unsedated colonoscopy for colorectal cancer screening. Medline data from 1966 to 2009 were searched to identify relevant articles on the subject. Data were summarized and co-authors provided critiques as well as accounts of unsedated colonoscopy for screening and surveillance. Diagnostic colonoscopy was initially developed as an unsedated procedure. Procedure-related discomfort led to wide adoption of sedation in the US, although unsedated colonoscopy remains the usual practice elsewhere. The increased use of colonoscopy for colorectal cancer screening in healthy, asymptomatic individuals suggests a reassessment of the burden of sedation in colonoscopy for screening is appropriate in the US for lowering costs and minimizing complications for patients. A water method developed to minimize discomfort has shown promise to enhance outcomes of unsedated colonoscopy. The use of scheduled, unsedated colonoscopy in the US appears to be feasible for colorectal cancer screening. Studies to assess its applicability in diverse practice settings deserve to be conducted and supported.

Porcine epidemic diarrhoea (PED) is a contagious enteric disease of pigs caused by a coronavirus. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (PEDV). The DAS-ELISA was compared with RT-PCR in the examination of 506 specimens collected during 2006-2007 from pigs originating from different farms located in the Po valley. Both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. The correlation between the two methods was higher when testing faecal samples (K=0.97, 95% CI: 0.94-1.00) than testing intestinal samples (K=0.62, 95% CI: 0.35-0.89). The use of ELISA technology provided an efficient and effective mean of evaluating the presence of coronavirus PED antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A lateral flow device (LFD) for the detection of foot-and-mouth disease virus (FMDV) of the SAT 2 serotype was developed using a monoclonal antibody (Mab 2H6). The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia: 305 positive for FMDV type SAT 2 from suspected cases of vesicular disease collected from 30 countries and 1002 samples shown to be negative for FMDV type SAT 2 collected from 67 countries between 1968 and 2008. The diagnostic sensitivity of the LFD for FMDV type SAT 2 was higher at 88% compared to 79% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 100% for the ELISA. The device recognized FMDV strains of wide diversity within the FMDV SAT 2 serotype and gave a superior performance for their detection compared to the 1F10 LFD which had been developed previously and shown to perform less well for the detection of FMDVs of this particular serotype. Reactions in the SAT 2 2H6 LFD with the viruses of other FMDV serotypes and swine vesicular disease (which produces a clinically indistinguishable syndrome in pigs), did not occur. These data illustrate the potential for the LFD to be employed to complement the 1F10 device next to the animal in the pen-side diagnosis of FMD, for providing rapid and objective support to veterinarians in their clinical judgment of the disease and for specific confirmation of a FMDV type SAT 2 infection., (2009 Elsevier B.V. All rights reserved.)

Aim: To assess the patient-reported outcomes (PROs) of pancreatic neuroendocrine tumor (PNET) patients., Methods: Fifty-one consecutive patients (21 male, 30 female, 61.0 +/- 10.3 years) with proven PNETs were studied. An SF-12 questionnaire capable of exploring the physical (PCS) and mental (MCS) aspects of daily life was used. Four questionnaires were also used [12 items General Health Questionnaire (GHQ-12) for non-psychotic psychiatric disorders, State Trait Anxiety Inventory (STAI) Y-1 and Y-2 for anxiety and BDI-II for depressive symptoms] to explore the psychological aspects of the disease. Forty-four sex- and age-matched Italian normative subjects were included and evaluated using the SF-12, STAI Y-1 and Y-2 questionnaires., Results: Seven patients refused to participate to the study; they were clinically similar to the 44 participants who agreed to complete the questionnaires. PNET patients had a PCS score (44.7 +/- 11.0) were not significantly different from the norms (46.1 +/- 9.9, P = 0.610), whereas the MCS score was significantly lower in patients (42.4 +/- 13.0) as compared to the norms (48.2 +/- 9.8, P = 0.036). GHQ-12 identified 11 patients (25.0%) as having non-psychotic psychiatric disorders. The STAI scores were similar in the patients and in the normative population. Finally, BDI-II identified eight patients (18.2%) with moderate depression and 9 (20.5%) with mild depression whereas 27 patients (61.4%) had no depression., Conclusion: The PNET patients had a good physical but an impaired mental component of their quality of life; in addition, mild or moderate depressive symptoms are present in about 40% of PNET patients.

RNA helicase-like receptors MDA-5 but not RIG-I has been shown to be essential for triggering innate immune responses against picornaviruses. However, virus-host co-evolution has selected for viruses capable of replicating despite host cells antiviral defences. In this report, we demonstrate that RIG-I is degraded during encephalomyocarditis virus (EMCV) infection. This effect is mediated by both the viral-encoded 3C protease and caspase proteinase. In addition, we show that RIG-I overexpression confers IFN-beta promoter activation during EMCV infection, in MDA-5 knockout (MDA-5(-/-)) mouse embryo fibroblasts. This induction is followed by a strong inhibition reflecting the ability of EMCV to disrupt RIG-I signalling. Taken together, our data strongly suggest that during evolution RIG-I has been involved for triggering innate immune response to picornavirus infections.

The objective of this study was the development and the evaluation of competitive ELISA assays based on monoclonal antibodies for the detection of antibodies specific for neuraminidase type 1 (N1), 2 (N2) and 3 (N3) in avian sera. A total of 1450 sera from different avian species (854 negative, 185 positive to N1, 136 positive to N2, 219 positive to N3 and 56 positive to other N subtypes sera) were analysed in parallel by the three ELISAs. ROC analyses were performed to enable the selection of best cut-off values and estimation of diagnostic specificity and sensitivity. In addition, the correlation between the new developed ELISAs and the neuraminidase inhibition test was evaluated on a limited number of sera. The validation process of the three ELISAs proved excellent diagnostic performances, with very high specificity and sensitivity, ranging from 99.4 to 99.8% and from 97.6 to 100%, respectively in the three assays. The discriminating potential between antibodies elicited against homologous and heterologous N validates the test for use in "DIVA" assays, to distinguish between vaccinated and infected birds.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C(PRO)) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

The presence of visceral hemangiomas in the syndrome of multiple enchondromas and subcutaneous hemangiomas, also named Maffucci syndrome, is exceedingly rare; until now noncutaneous hemangiomas have been described in 4 patients: they were found in the tongue in one patient, in the oral cavity in the second patient, in both the oral cavity and the colon in the third patient, and in the spleen in the fourth patient. We report the first case of hemangioma localized in the liver in a patient with Maffucci syndrome; furthermore, due to the impossibility of carrying out an MRI to define the hepatic lesion, an ultrasonographic real-time perfusion imaging study with a contrast agent was performed.

Aims: To explore the quality of life in patients treated medically during the acute phase of pancreatitis as well as at 2 and 12 months after discharge from the hospital., Patients: 40 patients were studied. The etiology of the pancreatitis was biliary causes in 31 patients and non-biliary causes in 9; mild disease was present in 29 patients and severe disease in 11. 30 patients completed the two surveys at 2 and 12 months after hospital discharge., Methods: The SF-12 and EORTC QLQ-C30 questionnaires were used for the purpose of the study., Results: The two physical and mental component summaries of SF-12, all the domains of EORTC QLQ-C30 (except for physical functioning and cognitive functioning) and some symptom scales of EORTC QLQ-C30 (fatigue, nausea/vomiting, pain, and constipation) were significantly impaired during the acute phase of pancreatitis. There was a significant improvement in the SF-12 physical component summary, and global health, role functioning, social functioning, nausea/vomiting, pain, dyspnea, and financial difficulties (EORTC QLQ-C30) at 2 months after discharge as compared to the basal evaluation. Similar results were found after 12 months except for the mental component score at 12-month evaluation, which was significantly impaired in acute pancreatitis patients in comparison to the norms. The physical functioning of the EORTC QLQ-C30 at basal evaluation was significantly impaired in patients with severe pancreatitis in comparison to patients with mild pancreatitis., Conclusions: Two different patterns can be recognized in the quality of life of patients with acute pancreatitis: physical impairment is immediately present followed by mental impairment which appears progressively in the follow-up period., (Copyright 2009 S. Karger AG, Basel.)

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.

Nine viral diseases included in the World Organization for Animal Health list of notifiable diseases (former list A) were chosen for their contagiousness and high capacity of spreading to improve their diagnosis using new and emerging technologies. All the selected diseases--foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza--are considered as transboundary diseases, which detection causes the prohibition of livestock exportation, and, thus, it leads to high economical losses. The applied diagnostic techniques can fall into two categories: (i) nucleic-acid detection, including padlock probes, real-time PCR with TaqMan, minor groove binding probes and fluorescence energy transfer reaction probes, isothermal amplification like the Cleavase/Invader assay or the loop-mediated amplification technology and the development of rapid kits for 'mobile' PCR and (ii) antigen-antibody detection systems like simplified and more sensitive ELISA tests. Besides, internal controls have been improved for nucleic acid-detecting methods by using an RNA plant virus--Cowpea Mosaic Virus--to ensure the stability of the RNA used as a positive control in diagnostic real-time RT-PCR assays. The development of these diagnosis techniques has required the joint efforts of a European consortium in which nine diagnostic laboratories and an SME who have collaborated since 2004 within the European Union-funded Lab-on-site project. The results obtained are shown in this paper.

The widespread perception of the effectiveness of applying tests based on the detection of antibodies against foot-and-mouth disease (FMD) viral non-capsid proteins (NCPs) to assess virus circulation irrespective of vaccination triggered the demand for international standards to evaluate the comparative performance of the upcoming assays against the OIE Index test developed at the Pan American Foot-and-Mouth Disease Center, PAHO/WHO. To this end, a panel was developed composed of 34 cattle sera from animals with an unambiguous exposed/infected status, covering serotypes O, A and C, obtained either under experimental conditions or from the field in regions with different epidemiological situations. Reference values in the Index test and their reproducibility in other laboratories, data on stability as well as results in four other commercial kits and one in house test were obtained. The characteristics of the panel which comprise adequate preparation following international guidelines, a broad range of antibody reactivity, proper stability and the ability to assess comparative diagnostic sensitivity, make it suitable as a reference standard to evaluate if tests equivalent to the OIE Index method are used in support of FMD control programs and by trading partners, and also whether they maintain their standards of diagnostic performance.