Development And Initial Application Of Antigen-capture ELISAs For The Detection Of Equine Influenza Virus

Equine influenza virus (EIV) causes an acute respiratory infectious disease with international infectiousness, highly contagious, variable morbidity and low mortality in horses. With the success of Beijing Olympic Games and Guangzhou Asian Games, the horse-race is an emerging industry. The prevention and control work for the equine influenza in China has a long way to go when confronted with gradually expanded industry of horses and donkeys. Therefore, it is imperative to develop an assay that could be widely used for the routine diagnosis.For investigating the nucleoprotein (NP) of EIV A/equine/Xinjiang/07(H3N8) strain (XJ isolation), the NP gene amplified by RT-PCR was cloned into pMD-18T. After analysis by sequencing, the positive one was digested and subcloned into prokaryotic expression vector pET-30a to construct the recombinant plasmid pET30a-NP. The positive pET30a-NP was transformed into Rosetta (DE3), then the transformed bacteria were induced by IPTG and produced a recombinant protein of 64 ku mostly in soluble form which was subsequently purified by Ni+-column. The expressed protein and purified one were both reacted well with the positive serum against EIV, indicating that they had good antigenicity.The 4 weeks-old BALB/c mice were intraperitoneally immunized with the purified recombinant protein NP. Then the spleen cells of the immunized mouse were fused with SP2/0 myeloma cells according to standard procedures. Meanwhile, an indirect ELISA using the purified XJ isolation as antigen was developed to screen positive hybridomas. Consequently, three monoclonal antibodies (MAbs) named 2G11, 3E10 and 4A1 were produced. The results of Western blot , indirect immunofluorescence and indirect ELISA showed that the three MAbs could react with the recombinant NP specifically and the natural EIV well. All of them had no cross reactions with Japanese encephalitis virus, equine herpes virus-1/4 and equine arteritis virus.The rabbits were immunized with the purified XJ isolation and NP to produce the polyclonal antibodies against virus and NP respectively. Then four kinds of antigen-capture ELISAs were developed using the MAb 2G11 and the polyclonal antibody against virus or NP. The specificity of the optimized antigen-capture ELISAs was evaluated using EIV, Japanese encephalitis virus, equine herpes virus-1/4 and equine arteritis virus, resulting in only EIV specimens yielding a strong signal. Compared with hemagglutination test, the sensitivity of each method was higher than that of the later.The fourth one which used polyclonal antibody against NP as capture antibody and MAb 2G11 as detecting antibody could detect avian-like strain A/equine/Jilin/89(H3N8) (JL isolation),European lineage strain A/equine/Qinghai/94(H3N8) (QH isolation) and A/equine/Miami/63(H3N8) (Miami isolation) in higher titers than the other three ones. Meanwhile each had cross-reactivity with H7N7 subtype.Three seronegative foals were exposed to an aerosol of EIV XJ isolation. Viruses from the nasal swabs collected for 10 consecutive days after challenge were detected from days 3 to 7 post-infection using the fourth antigen-capture ELISA, with results confirmed by virus isolation and multiplex RT-PCR, indicating a suitable method for rapid detection of EIV.