Strategy Research Of Labeling Envelope And Viral Ribonucleoprotein Complexes Of Influenza A Virus With Quantum Dots

Influenza A virus (IAV) caused seasonal influenza epidemics and sporadic influenza pandemics in history have exerted a disastrous effect on human civilization. At present, vaccination and antiviral medication are main routes to prevent and treat the diseases caused by IAV. However, under selective pressure, there are more and more virus strains that are resistant to antiviral drugs today. This tendency warms us "no action today means no cure tomorrow", thus developing new antiviral drugs for IAV becomes extremely urgent. On the basis of this situation, it is necessary and urgent to understand the infection mechanisms of IAV in depth, which could provide novel drug targets and theoretical basis for the research and development of new antiviral drugs.Single-virus tracking technique, which can enable us to real-time monitor individual virus infection in live cell, is a powerful tool of investigating the infection mechanisms of viruses. To visualize the complete process of IAV infection through single-virus tracking technique, one of the prerequisites is to label both of the envelope and viral ribonucleoprotein (vRNP) complexes of IAV with some bright and photostable beacons such as quantum dots (QDs) via some reasonable and reliable labeling strategies. Nevertheless, so far, limitations still exist in the conventional strategies for labeling of surface components of virus, as well as no strategy has been developed for labeling inner-components of virus. On the basis of the current status, this thesis aims to overcome the limitations in the conventional strategies for labeling of surface components of virus with QDs and fill gaps in strategy for labeling inner-components of virus with QDs.We devised a hydrazine-aldehyde reaction based strategy to label the envelope of IAV with QDs through the conjugation of aromatic aldehyde modified QDs to aromatic hydrazine derivatives modified H9N2. This strategy has advantages of high selectivity, high efficiency of labeling that more than 90% viruses have been labeled, and maximum retention of both infectivity of H9N2 and fluorescence properties of QDs. Moreover, in comparison with the widely used biotin-streptavidin based strategy, the hydrazine-aldehyde based strategy has a little bit higher efficiency of labeling and better retention of virus infectivity. More importantly, QDs labeled viruses obtained through hydrazine-aldehyde based strategy can avoid the interference of endogenous biotin, which is good for ensuring the reliability of information obtained from single-virus tracking. Because abundant exposed amino groups exist on the surface of various viruses, this hydrazine-aldehyde based strategy is also applicable to other viruses such as PrV.Then, to achieve the ultimate goal of labeling both of the outer- and inner components of IAV, we devised a convenient strategy to label the vRNP complexes in IAV. Briefly, by using of electroporation techniques, green fluorescent QDs (GQDs) labeled nucleoprotein antibodies (NPAb) can enter into H1N1, then GQDs-NPAb can recognize and link to the vRNP complexes inside the H1N1. The feasibility of this strategy has been confirmed by a series of experimental results. Through the optimal experiments in the range of reverse electroporation, pulse intensity of 750 V/cm, pulse duration of 20 ms, pulse number of 1 time and 20 nM GQDs-NPAb have been determined as the optimal conditions of labeling. Under the above conditions, the actual efficiency of labeling is about 34.72%, and the QDs labeled H1N1 retains 92.59% infectivity. Moreover, because different subtypes of IAV have similar structure and components, this labeling strategy is also applicable to other subtypes of IAV, e.g. H9N2.On the basis of the above works, we successfully obtained inner-and outer-components dual-labeled H1N1 through labeling of vRNP complexes with GQDs under optimal conditions followed by labeling of the envelope with red fluorescent QDs (RQDs) via hydrazine-aldehyde based strategy.5 nM RQDs has been determined as optimal concentration through optimal experiments. Under optimal conditions, the actual efficiency of dual-labeling is about 10.89%, and the dual-labeled H1N1 retains 93.25% infectivity. Because both of the two strategies used in dual-labeling have good university, this dual-labeling strategy is also applicable to other subtypes of IAV such as H9N2.