Study On Morphogenesis Of Rabies Virus SRV9Strain In Murine Primary Neuron Cells

To investigate the pathogenicity and replication of rabies virus SRV9strain in murine primary neuron cells, and to understand the morphogenetic of the rabies virus in the nerve cells, we isolated and cultured primary murine neuron cells. After morphological observation of the separated primary murine neuron cell, we infected the primary neuron cells with SRV9strains of the rabies virus. The morphological changes of SRV9strains in primary neuron cells were observed with direct immunofluorescence, immunohistochemistry, transmission electron microscopy and laser scanning confocal microscope. The results showed that the primary murine neurons cells can be used as a cell model for the study of rabies virus infection in vitro.The mouse brain tissue was digested and separated into cell suspension with trypsin. The primary neuron cells were cultured with DMEM medium containing10%horse serum and then identified by morphological observation. The methods of direct immunofluorescence and immunohistochemistry were used to detect whether the virus infected primary neuron cells. On this basis, rabies virus SRV9strains (100TCID50) was inoculated with primary neurons cells. The virus growth and proliferation in the cell were detected using direct immunofluorescence and laser scanning confocal microscope at3d、4d、5d respectively after infection. Under the same conditions, preparation of the ultra-thin slice of primary cultured neuron cells infected with rabies virus and observation of the morphogenesis of rabies virus in primary murine neurons under transmission electron microscopy.The results showed that the primary murine neuron cells could be cultured in vitro. The cells could differentiate into multi-polar nerve cells and form a network with adjacent nerve cells. The synaptic connections between neurites of primary mouse nurone cells were observed at5d of cultivation. After cultivation of6to12days, the nerve cells exhibited best status, however gradually degenerated and died when cultivated in vitro for25to35days. The rabies virus in primary neuron cells could be initially detected at2d and the viral antigen reached the maximum at5d to7d by the direct immunofluorescence and immunohistochemistry experiments. Under the same conditions, the viral antigen localizated in the cytoplasm and neutite of the nerve cell soma after infection at3d to5d, but the virus particle was not observed in the nucleus using direct immunofluorescence and laser scanning confocal microscope. The results by ultrathin sections showed that the virus replicated and synthesize protein in the cytoplasm after infection of cells, and then formed capsule by vacuolar membrane and membrane budding after cell disruption.Conclusion:Primary murine neuron cells could be isolated and cultivated under the special condition. SRV9strain of the rabies virus can infect primary murine neuron cells, and replicate in primary cultured neurons. Therefore, the primary murine neurons cells can be used as a cell model for the study of rabies virus infection in vitro. This experiment lay the foundation for study of the pathogenesis of rabies virus.