Screening And Identification About The Differentially Expressed Genes Related To Cytoskeleton And Transmitters Of Neuron After Street RABV Infection

Rabies is a fatal zoonosis disease, which characterized as fatal encephalitis of thecentral nervous system in humans and animals. The obvious clinical sign ofneurological symptoms always related to street rabies virus infection. Even rabies isan ancient disease, So far, its pathogenesis remains largely unknown. It is well knownthat cytoskeleton plays an important role in both intracellular transport and cellularmorphology, so a street rabies virus was screened and expression of glycoprotein（G）gene and antigens distribution in the different areas of brain were determined,moreover，differentially expressed genes related to cytoskeleton and transmitters ofneuron in hippocampus were analyzed, and these might provide some data foreilluminating dysfuction in nervous system.In preliminary studies, glycoprotein genes of two fixed strains (CVS3aG strains)and two street rabies virus (SX, PB3) were sequenced. The homological analysisshowed that deduced signal peptide of glycoprotein was100%between the two Streetstrains, and84.2%was of the fixed strains, and78.9%and73.7%was among thestreet strain and CVS or3aG, respectively. The signal peptides of the street strain wereexactly the same as Street strains by NCBI Indexed from China in recent years, andhomology of the signal peptides of Street strains and routinely used vaccine strainsranged from68.4-84.2%,even predicted secondary structure and hydrophobic valuesin signal peptide of glycoprotein of street rabies viruses were significantlydifferentiated from the routinely used vaccine strains and fixed strains of rabiesviruses. And a substitution at247glycosylation site in G was observed, whichpresented as N（Asn）to→D（Asp）mutation. Moreover,20C57BL/6mice were randomly divided into two groups. Oneswere infected with street rabies virus PB3strain by intracranial inoculation at a doseof10MICLD50/30μL, another were inoculated with the same volume of C57BL/6mouse brain suspension. When the infected mice got close to agonal stagesequentially, expression levels of G gene in different brain regions of mice weremeasured by fluorescent quantitative PCR（Q-PCR）,and the virus distributions weredetected by immunohistochemistry. The order of G gene copies was cerebral cortex>hippocampus, brain stem> cerebellum> thalamus>olfactorybulb. which wascoincident with RABV distribution by immunohistochemistry.then in the agonal stage of infected mice, hippocampus tissues were gathered,and40genes related to the cytoskeleton and neurotransmitters were determined byQ-PCR. The results showed that the changes of some gene in vitro and vivo wereconsistent, these genes included Stmn1(↓), katna1(↑), MYLK (↑) gelsolin (↑), kif5b(↓), APC (↑) klc1(↓), and GSK3beta (↑). stmn1, katna1, APC, Gsk3β. It is wellknown that stmn1, katna1, APC, Gsk3β regulate stability of intracellular microtubules,MYLK damages intracellular F-actin;and kif5and klc1hindered transport of neuronalfactors and vesicles and MLCK regulate actins and releasing of neurotransmitters.some ultra-structure changes in the neurons of hippocampus were observed underelectron microscope, which presented as swelling of mitochondrion, disappearancemitochondrial cristaes, hyperfunction of Golgi complex, expansion of perinuclearcisterna.Based on the results, it implicated that the rabies pathogenesis might attribute tostructure changes and destruction of the cytoskeleton and abnormality of transmissionand releasing of neurotransmitters in neurons,all these may be eilluminated to thedysfunction associated to street rabies virus infection.