| Avian influenza, caused by the avian influenza viruses(AIV), is the main infectious disease of poultry industry in China. The symptoms aftering infection may be a slight respiratory disease, reductions in egg production or even the death. At the same time, AIV has the ability to transmit from bird to human and seriously threaten the security of public health. So it is extraordinary important to research the virus in depth.AIV has a variety of different subtypes, what’s more, the pathogenicity among different subtypes is obvious difference. So to determine the subtypes of AIV quickly and accurately is the prerequisite for taking effective measures to prevention and control of avian influenza. AIV is further divided into different subtypes on the basis of antigenic variation transmembrane glycoproteins: haemagglutinin(HA) and neuraminidase(NA). The HA subtype is usually determined quickly and accurately by haemagglutination inhibition(HI) tests. While neuraminidase isolates are antigenically characterised by neuraminidase inhibition(NI) assays, the process of which is complex and time-consuming. In order to meet the actual needs, we try to develop a new diagnostic test for NA as effectively as possible. In this study, we used baculovirus expression system to generate recombinant baculovirus which express different NA subtype of AIV in insect cells and ultimately obtained N1-N9 neuraminidase proteins successfully. The recombinant proteins were analyzed by western blot and the results indicated that they had good reactivity with antiserum and all of them were enzymatically active. What’s more, we have laid a foundation for futher study of the ELISA assay for NA subtype after exploring the reaction conditions carefully.Non-structural protein 1(NS1) of AIV, the most important viral regulatory factors, facilitates viral replication through interacting with a variety of host proteins. In order to identified cellular protein interacting with NS1, we applied immunoprecipitation(IP) to screen host factors after A549 cells infected with highly pathogenic H5N1 avian influenza virus(A/duck/Guangdong/ S1322/2010). After mass spectrum(MS) identification, annexin A2(ANXA2) protein was suspected as our target.The interactions were further confirmed by co-IP, and the results of confocal laser scanning microscopy assay indicated that the NS1 protein associated with ANXA2 in cytoplasm. What’s more, we have demonstrated the knockdown of ANXA2 with siRNA greatly inhibited the replication of H5N1 AIV, and contrary to the results of overexpression of ANXA2. It is suggested that ANXA2 may accelerate the replication of AIV and the interaction between NS1 and ANXA2 may assist NS1 to promote the viral multiplication. At the same time, MS also identified Apoptosis-inducing factor 1, mitochondrial(AIFM1) may interacted with NS1 protein. We used co-IP to confirm the interaction, and we also indicated AIFM1 interacted with the effective domain(ED) of NS1 directly by Pull down assay. And the results of confocal laser scanning microscopy assay indicated that the NS1 protein associated with AIFM1 in cytoplasm. Taken together, we firstly confirmed that NS1 interacts with ANXA2 and AIFM1 respectively and provided a new perspective to further research the function of the NS1.In this study, we make a research on practical application and replication mechanism of AIV respectively. Our work is the basis of future establishment of detection of NA quickly and understanding of AIV’s replication deeply. This study benefits to current research of AIV. |
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