RNA Interference Against Rabies Virus In Vivo And In Vitro

RNA interference (RNAi) refers to the small dsRNA-guided gene silencing phenomenon conserved in a wide range of eukaryotic organisms from plants to mammals. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression, the prevention of viral infection and the control of gene transposition. RNAi in target cells could be induced specifically and effectively by 21nt to 23nt short interfering RNA (siRNA) and has become a powerful tool to explore gene functions replacing the knock-out technique. With the progressions of the research about RNAi, some investigations have been performed successfully to inhibit the replication of viruses such as Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Dengue virus, Poliovirus, Influenza virus (IV), Foot-and-Mouth disease virus (FMDV), severe acute respiratory syndrome (SARS) coronavirus, and so on.Rabies is a lethal zoonotic infection disease induced by Rabies virus and its case fatality ratio (CFR) is 100 percent approximately. China is one of the most severe regions of rabies spread. According to the recommendations of WHO rabies experts committee, post-exposure treatment (PET) must be taken as soon as possible after a bite or a scratch occurred. Immunoglobulin is one of the regents in PET, but the zoogenous immunoglobulin is a kind of allergen, and the products made from human blood are infective agents of some body fluid transmitted diseases. The discovery of RNAi mechanism and the related explorations in antiviral therapy established a new direction of rabies and rabies virus. In this thesis, the replication of rabies virus was inhibited by RNA interference in vivo and in vitro experimentally in order to accumulate essential data of the application of RNAi in the researches of rabies virus genomic function and the PET of rabies.Firstly, thirty target sequences and a control sequence aimed at the rabies virus mRNAs of the construction proteins were designed, the insert DNA sequences were synthesized respectively and their expression vector were constructed based on the plasmid pSilencer 2.1-U6 Hygro (Ambion). Twenty two cell strains expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into the BHK-21 cell line and screened under the pressure of Hygromycin B (300μg/mL). These cell strains in a 24-well tissue culture cluster were infected by 1000 TCID50 rabies virus CVS-11 strain respectively, and 48h later, the virus replication were detected and evaluated by directed immunofluorescence assay (DFA). The results showed that the inhibition ratio of the virus replication were 98.9%～3.3% in different cell strains and the most two noticeable strains were BHK-N19 (98.9%) and BHK-G69 (94.6%). The RT-PCR amplifying the nucleoprotein gene fragment indicated that the products were inverse correlation to the virus inhibition ratio.Some siRNAs aimed at the target sequences N-19 and G-69 were synthesized and transfected into BHK-21 cells induced by RNAiFect (Qiagen), 48h later, the virus infection ratio were 11.0%和17.4% compare with the control ratio of 82.6%, and the virus inhibition ratio were 86.7%和78.9%. The effects of RNAi were coincidence between the different transfectional buffers such as MEM and EC-R (Qiagen). The later rabies virus infected the cells transfected by siRNA N-19, the more obviously rabies virus replicated. The inhibition ratios decreased from 86.6% to 25.6% when the virus infected the cells at 24h to 96h after the transfection. The growth curve of rabies virus in the cells transfected by siRNA-N19 was milder than the control.Mouse model of rabies infection was made by injecting 50LD50 rabies virus CVS-24 into a hind lamb intramuscularly and then treated by the injections of plasmids or synthesized siRNAs as pre-exposure prophylaxis (PEP) or post-exposure treatment (PET). The results showed that only the reagents aimed at the glycoprotein mRNA were effective. 80% (16/20) and 55% (11/20) of the challenged mice survived after the PEP and PET with 100μg plasmid p2.1-G69, and the incubation period were prolonged significantly (P<0.01). siRNA-G69 could rescue about 20% (4/20) mice after PET, and the incubation period were prolonged significantly (P<0.05) too. A direct correlation present between the survival ratios and the dosage of plasmid p2.1-G69 less than 100μg in PET. A therapeutic trial indicated that the plasmid p2.1-G69 was inefficacy to rabies at prodromal stage.