The M2e protein is low in copy number on the virus particle, but it is abundantly expressed on the surface of an infected cells [3, 21]

The M2e protein is low in copy number on the virus particle, but it is abundantly expressed on the surface of an infected cells [3, 21]. also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development. Introduction Matrix protein 2 (M2) of avian influenza virus (AIV) is a 97 amino acids (aa) protein encoded by RNA segment 7 of the influenza A TAK-632 virus (IAV) [1]. It is translated from spliced mRNA and shares a common start codon with the matrix 1 (M1) protein and the first nine aa, while the remaining 88 aa continues at the second (+1) open reading frame [1, 2]. In its native state, M2 is a Tnxb homotetrameric type III integral membrane protein composed of three domains; namely, a 54 aa cytoplasmic domain located in the viral envelope or cytoplasmic membrane of infected cells, a 19 aa transmembrane domain, and an N-terminal 24 aa ectodomain (M2e) which is exposed on the surface of the virus infected cells and on the viral particles [1, 3C5]. In the infected cell the M2 protein forms an ion channel which is vital for viral genome delivery into the host cell during virus entry [2, 3, 5C8]. Briefly, M2 ion channel activity is activated by acidification of virus-containing endosomes after internalization of the virus particle into the host TAK-632 cell via clathrin-dependant and Cindependent mechanisms [9, 10]. Amino acids 1C9 of the M2e protein are highly conserved across AIV strains, while minimal aa variation is observed for residues 10 to 24, making it an attractive target for AIV universal vaccine development [2, 11C20]. The M2e protein is low in copy number on the virus particle, but it is abundantly expressed on the surface of an infected cells [3, 21]. This differential epitope density between infected cells (high) and a mature virion (low) [15, 22] is the key feature for its recommendation as a marker for differentiating infected animals in vaccinated population (DIVA), a strategy used in AIV surveillance [23, 24]. The sensitivity and specificity of M2e-based DIVA have been demonstrated in our previous works [25C27]. This raised our interest towards the potential use of M2e TAK-632 in a competitive enzyme-linked immunosorbent assay (ELISA) format as a surveillance tool for AIV infection. The principle of competitive ELISA lies in the ability of the test subject antibody (e.g. chicken) to inhibit competitor antibodies, usually produced in rabbit or mouse, from binding to the target antigen. Hence, it is important for the competitor antibodies to react with the same viral epitopes as the antibodies produced by the test species. This ELISA structure continues to be showed for the nucleoprotein of AIV effectively, which provides shown to become applicable and reliable for multispecies surveillance [28C30]. Nevertheless, M2e-based competitive ELISA is normally a better choice DIVA check for an AIV security tool, in the extremely pathogenic AIV H5N1 endemic countries specifically, where chicken vaccination using inactivated AIV is normally practiced. It really is recognized that because of distinctions in the germline gene repertoire in various types, followed by distinctive systems for affinity and era maturation of antibodies, antigenic determinants acknowledged by a host may differ from one types to some other [31C33]. Earlier research on M2e proteins for vaccine advancement have reported many antigenic determinants discovered by anti-M2e antibodies isolated from rabbit, mouse and individual [20, 34, 35]. Generally, the M2e epitopes regarded were situated in the spot that span in the N-terminal to the center area of M2e, and differ long from 5 residues (2SLLTE6) [35], up to 15 residues 2SLLTEVETPIRNEWG16 [20, 34]. Right here, we explain epitope mapping using anti-M2e antibodies from poultry, rabbit and mouse to recognize the M2e antigenic determinants for every antibody group, and to measure the most suitable pet way to obtain anti-M2e antibodies in M2e-based competitive ELISA as a sophisticated DIVA check for.