After three washes, an HRP-conjugated goat anti-mouse immunoglobulin antibody (produced in-house) was added at dilution 1/500 in dilution buffer (PBS with 0
After three washes, an HRP-conjugated goat anti-mouse immunoglobulin antibody (produced in-house) was added at dilution 1/500 in dilution buffer (PBS with 0.05% Tween 20 and 1% yeast extract) to each well and incubated again for 1 h at 37C. 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 Protopanaxatriol WNVs tested and good competition for antigen binding in ELISA assessments 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 computer virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid switch at E307 (Lys Glu) in the E protein gene, whereas 4D3 variants recognized Protopanaxatriol mutations encoding amino acid changed at E276 (Ser Ile) or E278 (Thr Ile). 3B2 and 3D6 mapped to a region around the lateral surface of domain name III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 acknowledged a novel specific neutralizing epitope on domain name II of E protein that has not previously been explained 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 name II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization. Keywords:West Nile computer virus, Monoclonal antibody, Epitope == Background == West Nile computer virus is an arbovirus member of the Japanese Encephalitis computer virus (JEV) serocomplex of the genusFlavivirusof theFlaviviridaefamily. WNV contamination is one of the most common arboviral infections and can cause encephalitis in humans. Its transmission cycle entails mosquito-vectors (mainlyCulex spp.) and birds as amplifying reservoirs, but a wide variety of vertebrate species, including reptiles, amphibians and mammals, such as equines and humans, are also susceptible to contamination [1]. The WNV genome is made up of a single stranded positive-sense RNA molecule that encodes three structural proteins (capsid (C); pre-membrane (prM); and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) [2]. The envelope E protein is the major surface protein of flaviviruses and the primary immunogen that plays a central role in computer virus attachment and access into a cell via membrane fusion [3]. Crystallographic analysis reveals that this E glycoprotein Protopanaxatriol of flaviviruses folds into three unique structural domains (I, II and III) [4-6]. In particular, domain name III of WNV E protein (DIII) is the putative receptor-binding domain name and is an important target for neutralizing antibodies and in vivo Protopanaxatriol protection [7-11]. The recent outbreaks of West Nile Disease in humans and horses in Europe and the spread of the computer virus from North through South America during the Splenopentin Acetate last decade suggest that the epidemiology of this contamination is evolving. In the Mediterranean basin, outbreaks of WNV contamination in recent years have been reported in France (2004 and 2006), Italy (2008, 2009) Morocco (2010), Spain (2010) and Greece [12]. WNV was previously considered an amazing agent, while it is now regarded as an emerging problem for both human and veterinary public health. These outbreaks have stimulated research into computer virus detection and characterization, underlining the need for quick assays. Although many methods have been developed for WNV diagnosis, it is generally hard due to the considerable antigenic cross-reactivity among flaviviruses, especially in geographic areas where two or more of these viruses are present causing sequential infections [13]. It has recently been shown that WNV and Usutu computer virus (USUV) have comparable transmission cycles, with overlapping geographic distributions [14,15]. In this context, MAbs having strong and specific reactivity to WNV antigens are the most suitable choice for the development of standardized diagnostic tools. The purpose of this study was to characterize a panel of monoclonal antibodies produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. The results suggest the applicability of these MAbs to numerous analytical methods for WNV diagnosis allowing the.
