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The Preliminary Research about Pseudorabies Virus Transporting Along the Neurons Axon

Author: YinZuoZuo
Tutor: LiuZhengFei
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Pseudorabies virus recombinant tracer viruses DRG primary culture microfluidic chamber system selective filter
CLC: S852.65
Type: Master's thesis
Year: 2013
Downloads: 1
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Abstract


Pseudorabies virus (Pseudorabies virus, PRV) belongs to subfamily of alphaherpesvirus, which can infect peripheral nervous system to establish a long latent infection, make the host lifelong carry the virus. PRV virus particles from inside to outside of have four layers:double-stranded DNA, capsid, tegument proteins, envelope proteins, the tegument proteins are divided into the inner layer of the tegument proteins and the outer tegument proteins, the inner tegument proteins are generally associated with capsid proteins, while the outer tegument proteins generally connected with the cytoplasmic domain of the membrane proteins. The process of the virus invading the neurons is very similar to that found in epithelial cells. First, at the end of axons of the peripheral nervous system the virus envelope proteins fuse with the cell membrane, and then the nucleocapsid retrograde transport to the nucleus of the cell body along the axonal microtubule. While the inner tegument proteins (UL36, UL37) and US3retrograde transport to the nucleus with nucleocapsid. However, the mechanism of the virus particles anterograde transport from the neuron cell body to the distal axon is much controversy, some researchers believe that the mature virus assembled at the neuronal cell bodies, then the mature virus particles transport to the axon terminals along the axonal microtubule to release. Others think that the virus nucleocapsid and envelope proteins were transported to the axon terminals separately along the axonal microtubule, and then at the distal of the axon assemble to release.Lately researchers found there is a selective filter that composed of an analytic G-and F-actin-dependent structure found in the cytoplasm at the axon initial segment (AIS) of hippocampus neuron diffusion of macromolecules and transport of vesicular carriers into the axon. It works as a filter screen that preventing macromolecules (70KD) passing through between the axons and cell bodies, but except small molecules (lOkD) and some membrane protein relying on specific motor protein. Further research found that the strength of the driving force of the motor proteins, and membrane proteins-motor protein complex impact on membrane proteins passing through the selective filter. So whether selective filter impact the PRV proteins getting into the cell body along the retrograde axonal transport process?Based on the above background, four recombinant tracer virus PRV VP16-EGFP, PRV UL36-EGFP, PRV EGFP-VP26, PRV gB-RFP were constructed in our experiment. Then how the tracer virus transport in the axon was observed. Primary chicken dorsal root ganglion was cultured in microfluidic chamber system in vitro, infected with the tracer virus at the axonal compartment, then the virus invaded into cell bodies or not was observed under confocal microscope. The main work including:1. Recombinant virus constructionIn this study, we tried to insert green or red fluorescent protein into the C terminal or N terminal of PRV capsid protein VP26(UL35), the inner tegument proteins VP1/2(UL36), the outer tegument protein VP16(UL48) and the envelope protein gB (UL27) to build PRV VP16-EGFP, PRV UL36-EGFP, PRV EGFP-VP26, and PRV gB-RFP recombinant tracer viruses. Firstly, PRV UL35, UL36, UL48, UL27gene upstream and downstream homology arms were successfully cloned from PRV-Ea genome. At the same time, we amplified EGFP and RFP gene respectively from pEGFP-Cl and pRFP-Cl plasmid, then these homology arms genes and fluorescent protein gene were connected into pcDNA3.1plasmid in turn to construct recombinant transfer plasmids. By liposome co-transfection and through several rounds of Plaque screening and purification, we obtained four kind of recombinants. Comparing the proliferative properties of these recombinant viruses with wild virus by plaque size and one-step growth curve, we found recombinant virus plaque was significantly smaller than the wild type, however there was no difference between the value-added curve.2. The primary culture of DRGDorsal root ganglions (DRG) were isolated from chick embryo hatching about8-12days under a dissecting microscope, direct inoculate in the serum-free medium (NGF2.5S,20ng/ml) for explant culturing about7-10days for observation. The same way to obtain the spinal cord dorsal root ganglion, then digest with0.125%trypsin into single cells, first seeded in the culture medium, after24hours replaced with feeding culture medium, To inhibit growth of non-neuronal cells, cytosine β-D-arabinofuranosidein was added at the third day, after48h replaced with fresh culture medium and cultured for7-10days to observe. The cultured cells test by indirect immunofluorescence verification is indeed the chick embryo dorsal root ganglion cells. We observe the tracer virus transport in neuronal axons, the proteins of virus transport in axonal bidirectional, and transport by leaps and bounds.3. The establishion of the microfluidic chamber systemTo establish chick embryo primary dorsal root ganglia (DRG) neuron cultures in a microfluidic chamber system, neurons planted in the soma compartment (S compartment), axons extending through the microgroove to the neurite compartment (N compartment). Four recombinant tracer viruses were inoculated in the neurite compartment for2hours, observe whether the fluorescent-labeled proteins can invade into the cell body with laser scanning confocal microscope. we calculate:The VP16-EGFP fused protein mobility can be observed in the axons of the neurite compartment and the microgroove, but can’t invade into the cell body of.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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