A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk

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

Relationship between neutralizing and opsonizing monoclonal antibodies against foot-and-mouth disease virus.

Previous studies demonstrated that polyclonal antibodies against foot-and-mouth disease virus (FMDV) generated by vaccination can mediate immune functions not only through virus neutralization but also through promoting virus uptake by macrophages and dendritic cells that are otherwise resistant to FMDV infection. This causes abortive infections resulting in activation, enhanced antigen presentation but also cell death. Here we report the use of RAW264.7 cells representing a murine macrophage cells line to characterize opsonizing functions of a collection of monoclonal antibodies (mAbs) against FMDV O and A serotypes. We demonstrate that all neutralizing immunoglobulin G isotype mAbs are able to opsonize FMDV resulting in increased cell death of RAW264.7 cells. In contrast, neutralizing IgM antibodies did not possess this activity. Opsonization was observed with broader reactivity within the serotype when compared to neutralization. Importantly, the anti-O serotype D9 mAb reacting with the continuous epitope within the G-H loop of VP1 that contains the RGD binding site of FMDV, opsonized several FMDV serotypes despite its restricted neutralizing activity within the O serotype. Furthermore, by generating RAW264.7 cells expressing bovine CD32, an easy-to-use cell-based assay system to test for bovine antibody-dependent enhanced infection of FMDV was generated and tested with a collection of sera. The data indicate that opsonizing titers correlated better with vaccine dose when compared to neutralizing titers. On the other hand, neutralization and opsonization titers were similar predictive of protection. We conclude that low avidity interactions are sufficient to mediate Fcγ receptor-mediated immune functions that could contribute to protective immune responses against FMDV

Combining multiple assays improves detection and serotyping of foot-and-mouth disease virus. A practical example with field samples from East Africa

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%

Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains

Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-gamma as a Key Player of the Enhanced Innate Immune Response in Lungs

Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-gamma) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-gamma.IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-gamma gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases

West Nile virus: characterization and diagnostic applications of monoclonal antibodies

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Eradication of Swine Vesicular Disease in Italy

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

Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

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