| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 90 === Enterovirus 71 (EV71), the newest member of Enteroviruses (Picornavirudae), has caused several large outbreaks including severe neurological diseases in children in recent years. Various viral isolates, including a tissue culture-adapted laboratory strain, have demonstrated different phenotypes such as growth rate in tissue culture and varying degrees of virulence in newborn mice. Genetic analysis of these viruses revealed differences in only a small percentage of nucleotides in both coding and noncoding regions. Mice infection models were attempted unsuccessfully in adult inbred, outbred or immune-deficiency mice. However, in newborn mice of all strains tested, a laboratory-adapted strain, termed YN3, inoculate resulted in hind limb paralysis and eventual death within two weeks post viral challenge. In order to dissect the viral proteins that are essential to these observed viral phenomena, we constructed plasmids encoding the complete cDNA of these EV71 genomes. The RNA transcripts of the full length cDNA EV71 genome, derived from in vitro transcription, are fully infectious when transfected into Vero cells, i.e., virus particles may be recovered from the culture medium of the transfected Vero cells. Mutant and Chimera EV71 clones were than generated from these constructs to study targeted virus proteins and altered biological properties. One motif located on the N-terminus of VP1, which was identified by analyzing VP1 genes of 12 Enteroviruses using SOPMA method to predict secondary protein structures. This motif contains helix, coil structure and proline residues upstream, which we prove to be essential to the virus infectivity by using the infectious cDNA system and it mutants. When the two prolines were replaced by leucines, no infectious virus particle could be recovered. However, when this leucine mutant was blindly passaged 5 times, a few plaques appeared. Sequencing analysis of these plaques showed reverse mutations of the leucine back to proline. Our data suggest that proline motif is essential for virus infectivity.
Since no effective antiviral agents are available, developing vaccines for primary prevention is considered to be the best choice among control strategies against EV71. We compared the inactivated virus vaccine with subunit vaccines - VP1 DNA vaccine or recombinant VP1 protein, in its ability to elicit maternal antibody and to provide protection against lethal infection of EV71 in suckling mice. Prior to gestation, all three groups of vaccinated dams possessed similar levels of neutralizing antibody. With a high challenge dose, suckling mice born to dams immunized with inactivated virus showed 80% survival. The subunit vaccines provided protection only at a lower challenge dosage, with 40% survival for DNA vaccine and 80% survival for VP1 protein. The cytokine profile produced by splenocytes showed a high level of IL-4 in the inactivated virus group, high levels of IFN- and IL-12 in the DNA vaccine group, and high levels of IL-10 and IFN-in the VP1 protein group. Overall, the inactivated virus elicited a much greater magnitude of immune response than the subunit vaccines, including total IgG, all 4 IgG subtypes, and T helper cell responses; these antibodies were shown to be protective against lethal infection when passively transferred to susceptible newborn mice. Our data indicated that inactivated virus is the choice of vaccine preparation capable of fulfilling the demand for effective control, and that VP1 subunit vaccines remain promising vaccine strategies that require further refinement.
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