Epitope Mapping And AC-ELISA Development Of Equine Infectious Anemia Virus P26

Equine infectious anemia(EIA) is a kind of animal infectious diseases, which is able to infect horses、donkeys、zebras and other equus. EIA can cause equine anaemia、thrombocytopenia、fever、wasting syndrome 、multiple organ failures and other symptoms. EIA was found by Lign é in 1843 in France for the first time. And this infectious disease is popular now around the world, such as America、Europe、the Middle East and South Africa and so on. The local animal husbandry、horse racing and related industries suffered huge economic losses because of it. During the 1950s~1960s, China also suffered many losses since epidemic EIA was severe in the middle and northwestern China. However, Rongxian Shen and other scientists developed the EIAV attenuated vaccine and EIA was effectively controlled then.Equine infectious anemia virus(EIAV) is the etiological agent of EIA. EIAV belongs to lentiviruses of Retroviridae family. As similar with other lentivirus such as human immunodeficiency virus(HIV) 、 simian immunodeficiency virus(SIV) and feline immunodeficiency virus(FIV), EIAV contain Gag、Pol and Env 3 major structural proteins and 3 non-structural proteins Tat、Rev and S2. Gag proteins, which is a polyprotein, can be processed to the matrix protein(MA) 、capsid(CA) 、the nucleocapsid protein(NC) and P9 protein eventually. These proteins function as parts of the virion structure.Capsid protein is also known as p26 protein, its function is to self-assemble the virus capsid structure. And its complete topological structure, namely the capsid of virus, packages of EIAV genome so as to ensure the efficient replication of the virus. Also as a main immunogenic protein of virus during the infection, p26 is highly conserved in structure and function among different EIAV strains. Moreover, p26 protein is one of the most abundant proteins in the virus particle.With 2 p26 monoclonal antibodies(MAb 9H8 and MAb 1G11) prepared in our Lab, this research used peptide-scanning technology, ultimately identifying MAb 1G11 and MAb 9H8 against linear epitopes of p26. MAb 1G11 targeted 21 amino acids of p26 protein C-terminal: 199KNAMRHLRPEDTLEEKMYAC218; while MAb 9H8 targeted 8 amino acids contained in the N-Terminal of p26: 73NLDKIAEE80. This result is helpful to analyze structure and function of p26 protein. Besides, it provides a basis for the analysis of epitope-based diagnostic methods and epitope-based vaccine design.According to State Council in the national medium-and long-term animal disease control program(2012-2020), the government plans to continue monitoring for EIA, especially intensive monitoring in the risky area of economic and recreational horses. And we could achieve the goal of eradication of EIA. According to the practical situation and national development project, to establish rapid and effective diagnostic methods for EIA is particularly urgent and important. Traditional diagnostic methods of EIAV is p26 protein antibodies detection with agar gel immunodiffusion test(AGID), also called the Coggins test. Although this method is highly specific, it lacks of sensitivity, which is particularly vulnerable for detection of chronic and non-obvious periods of EIA of clinical symptoms of infection. And viruses cannot be quantitated accurately, only by visualizing the sedimentation of the Antigen-antibody combination line to roughly assess the EIAV content. Plus, because of the long test cycles, it makes unlikely for practice of prevention timely and effectively and control of the epidemic.Linear epitopes identified in this study were aimed at EIAV p26 protein N-Terminal and C-Terminal. This makes it possible for establishment of antigen capture ELISA(AC-ELISA), because of characteristics of the targeted difference epitopes. Throμgh the using of EIAV(CMV 3-8) infectious clone or recombinant antigen p26 protein as standard antigens, MAb 1G11 and 9H8 as the capture antibody and enzyme labeled antibody, we established EIAV AC-ELISA to detect the clinical and experimental samples. With the EIAV AC-ELISA, the virus can also be quantitated, which is also helpful for quantification during viral replication and other related viral life cycle changes. It also contributes to EIAV quantitative analysis of EIAV antagonism between EIAV and host restriction factors like TRIM5-alpha, APOBEC3 family and Tetherin.In conclusion, with two monoclonal antibodies: MAb 9H8 and MAb 1G11 against EIAV p26 protein, this study used peptide-scanning technology to identify N-Terminal and N-Terminal linear epitopes of p26 protein. Because of recognition differences of MAb 1G11 and MAb 9H8, AC-ELISA was established to detect different EIAV viral strains. p26 linear epitopes identified maked it available to study structure and function of p26、epitope-based molecular label development and epitope vaccine designing; EIAV AC-ELISA can be applied to samples detection and related clinical practice、viral quantitative analysis and provide the platform and methodology of virus and virus-host interactions.