Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus

Centro de Investigación en Sanidad Animal (CISA)-INIA, Ctra Algete-El Casar, s/n, 28130 Valdeolmos, Madrid, Spain.
Journal of virological methods (Impact Factor: 1.78). 03/2011; 174(1-2):35-41. DOI: 10.1016/j.jviromet.2011.03.015
Source: PubMed


West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 μl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts.

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    • "West Nile Compac, INGENASA, Madrid, Spain) suitable for the detection of WNV antibodies in wild birds with low requirement of sample volume (Sotelo et al., 2011b). Neutralizing antibody titres were assessed by a standard virus-neutralization test (Sotelo et al., 2011b), using homologous virus as antigen. 2.7. "
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    ABSTRACT: Abstract West Nile virus (WNV) is a zoonotic pathogen which is maintained in an enzootic cycle between mosquitoes and birds; humans, equines, other mammals and some bird species are dead-end hosts. Lineage 1 WNV strains have predominated in Europe since the 1960's. However, in 2004 lineage 2 strains emerged in Hungary and Russia, respectively, spreading since then to a number of neighbouring countries (e.g., Austria, Greece, Italy, Serbia and Romania). Wild bird mortality is a hallmark of North American WNV outbreaks, a feature uncommon in Europe. This study aimed to compare the course of infection of lineage 1 (NY99) and lineage 2 (Austria/2008) WNV strains in the house sparrow, a bird species common in Europe and North America. House sparrows were inoculated with either NY99 or Austria/2008 WNV strains, or sham-inoculated, and clinical and analytic parameters (viraemia, viral load, antibodies) were examined until 14 days after inoculation. Although all inoculated sparrows became infected, no mortality or clinical signs were observed due to the infection. However, the magnitude and duration of viraemia were higher for NY99- than for Austria/2008- infected birds. The house sparrow proved to be a competent host for both strains, although the competence index calculated for NY99 was higher than for Austria/2008. Viral load in tissues and swabs was also higher in NY99-inoculated sparrows. In conclusion, the house sparrow is a convenient avian model for studying host competence of WNV strains. The observed differences between NY99 and Austria/2008 strains might have important epidemiological consequences for disease incidence and dispersal capacity.
    Veterinary Microbiology 08/2014; DOI:10.1016/j.vetmic.2014.06.005 · 2.51 Impact Factor
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    • "The iELISAs have the limitations of the requirement of highly purified coating antigen and non-specific background signals. In contrast, monoclonal antibody-based blocking ELISA (bELISA) can overcome these limitations and therefore it has been developed for the detection of antibodies, not only IgG isotype but also other immunoglobulin isotypes, to various viruses (Oem et al., 2007; Shearera et al., 2009; Heo et al., 2010; Huang et al., 2011; Sotelo et al., 2011). Monoclonal antibodies (mAbs) specifically recognize epitopes located in the C-terminal 268-amino acid region of avian HEV ORF2 protein have been produced in our laboratory (Dong et al., 2011). "
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    ABSTRACT: A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384-414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods.
    Journal of virological methods 04/2014; 204. DOI:10.1016/j.jviromet.2014.03.023 · 1.78 Impact Factor
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    • "Serum antibodies to WNV were detected by a commercially available epitope-blocking ELISA (Ingezym West Nile Compac, INGENASA, Madrid, Spain) suitable for the detection of antibodies to WNV in the serum of wild birds [26]. "
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    ABSTRACT: West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5-6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.
    Veterinary Research 03/2014; 45(1):33. DOI:10.1186/1297-9716-45-33 · 2.82 Impact Factor
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