Poliovirus I/Coxsackievirus B3 Chimeric Gene: The Construction And Primary Research Of Its Biological Character

Global polio eradication is the common goal of mankind. With the gradual decline of the acute flaccid paralysis (AFP) caused by wild poliovirus, vaccine-derived poliovirus (VDPV) is increasingly attracting the attention of people. The number of vaccine-associated paralytic poliomyelitis (VAPP), which is related to the chimeric virus formed by gene recombination betweenthe vaccine strain (V) and non-polio enteroviruses (NPEVS), has been growing up rapidly in recent years.Studying this kind of gene recombination will help us learn more about the mechanism of variation in sabin strain and the natural evolution trend of Enteroviruses. Therefore, it will benifit greatly the achievement of global poliomyelitis eradication.Based on the vector of pcDNA3, a cDNA copy of chimeric poliovirus type1 (PV1) genome was constructed, in which the P2 polyprotein encoding gene was replaced with the corresponding sequence from coxsackievirus B3, generating a new plasmid (pSCS) that can transcript and express in eukaryotic cell. After the transfection of this plsamid into KMB-17 cells, we found that the minority of these cells displayed altered morphology: some swelled ,turned round and detached from the culture plate ,others condenced and piled up likegrapes. Electron microscopic (EM) analysis showed that the number of lysosomes rose; the mitochondrions bulged and its critaes fractured, together with occurance of vacuole in mitochondrion matrix. The same modifications can also been found in the blind-passaged cells. Moreover, some virus-like particle (VLP) were observed in cytoplasm of two kinds of cell cultures. The result of immunoelectronmicroscopy (IBM) furtherly proved the existence of these particles. RT-PCR test illustrated that the chimeric plasmid transcripted in the transfected cells. Relying on the immunoprecipitation (IP) test ,we determinated the expression of viral proteins in transfected cells with different labeling time and found the VP0 protein band in samples at 8h,16h,24h and 36h. Data from the sucrose density gradient centifugation and liquid scintillation counting test suggested that the dynamic alternations of labeled viral proteins in pSCS plasmid- transfected cells were similar to the replicative and assembling process of poliovirus in control cells. Through the neutrilization test ,we also determinated the antibody from the mice vaccinated by the protein extracts of transfected cells and noticed that they protected the normal cells from the infection of PV1 to some extent while they didn't neutralize the challenge of CVB3.The above studying provided us further learning about the recombination phenomena of different virus.