Construction And Application Of Reverse Genetics Operating System Of Rabies Virus SRV9Strain

Rabies is a highly fatal zoonosis caused by rabies virus (RABV). Once theclinical symptoms have developed, human rabies is nearly100%fatal. RABV is asingle-stranded, non-segmented, negative-sense RNA virus. The genome encodesfive structural proteins. Recent years, with reverse genetics of negative-sense RNAvirus developing, several reverse genetics operating systems of RABV had beenbuilt. RABV vectors which were constructed by reverse genetic systems were usedfor developing novel vaccines and creating research tools in neuronal tracing and soon. RABV SRV9strain, screened from SAD strain by plaque-purify on BHK cells,is not pathogenic for several animals, but with good immunogenicity. Therefore,construction of reverse genetics operating system of RABV SRV9strain as wellselection novel safety and efficient RABV vaccines based on the system have asignificant meanings to rabies prevention and control. Besides, the system can bedeveloped as a new generation of RABV-based vectors against other infectiousdiseases.In order to analyze whole genome sequences of SRV9strain,13pairs ofsequencing primers were designed using software Primer Premier V5.0. Sequencingafter PCR one by one and connected to pEASY-Blunt vector. The results were thesame to the sequences published on line. Full-length genome of SRV9strain wasanalyzed by software DNAStar and divided into four parts according to the singlerestriction enzyme cutting site. The four parts were connected together based on theenzyme cutting site both in the two overlapping regions. Full-lengh genomiceukaryotic expression plasmids were generated based on vectors pCI orpcDNA3.1(+), namely pCI-SRV9or pD-SRV9. Meanwhile, utilizing PCR,3’ and5’of SRV9strain genome were inducted by hammerhead ribozyme (HamRZ) andhepatitis delta virus ribozyme (HdvRZ) respectively. Helper plasmids, expressing nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein (G) andlarge protein (L) of SRV9strain, were designed based on vectors pCI orpcDNA3.1(+), and namely pCI-N, pCI-P, pCI-G, pCI-L；pD-N, pD-P, pD-G, pD-L.During helper plasmids conduction, Kozak sequences were inducted before startcodon to enhance protein expression and extra stop codon was inducted followingstop codon to end transcription effectively.pCI-SRV9or pD-SRV9together with corresponding helper plasmids weretransfected into BSR cells using Liposome2000to rescue recombinant rabies virus.The amount of whole genome and helper plasmids N, P, L, G were2.5μg,0.625μg,0.3125μg,0.125μg,0.1875μg respectively. The results showed that both the twoplasmids expression systems could successfully rescue recombinant RABV.However, recovery efficiency of pD-SRV9(8/8) was significantly higher thanpCI-SRV9(3/30). The biological analysis of recombinantvirus in vitro suggested thatthere were no significant differences betweenthe recombinant SRV9and wild-typeSRV9(p>0.05).An exogenous gene expression component was introduced between the P and Mgenes of SRV9strain through PCR. According to different positions into whichcomponent introduced, two different vectors were designed. One vector waspD-SRV9-PM-eGFP, which exogenous gene expression componentPE-PS-BsiWI-PmeI was inserted between stop codon of P gene and transcriptionstop signal of P gene. Another vector was pD-SRV9-sPM-eGFP, which exogenousgene expression component PS-BsiWI-PmeI-PE was inserted between transcriptionstop signal of P gene and transcription initiation signal of M gene. PE and PS are thesequences which stimulant transcription stop signal (TGAAAAAAA) andtranscription initiation signal (AACACCCCT) of P gene of SRV9strain. The eGFPgene served as the reporting gene was inserted in the enzyme cutting sitesBsiWI-PmeI of vectors built above. The two kinds of full-length genomic eukaryoticexpression plasmids which contained exogenous gene expression component weretransfected into BSR cells with helper plasmids. The results showed that both thetwo vectors could successfully rescue recombinant virus expressing eGFP, and therewere no differences between the two vectors in recovery efficiency. There were no differences among the two recombinant RABV and the wild-type RABV in growthkinetics in vitro, either. This implied that the growth characteristics in vitro of therecombinant RABV would not be influenced by foreign proteins eGFP expressingcompared to the wild RABV.Here, we constructed the reverse genetics operating system of RABV SRV9strain which could express foreign proteins successfully. This allows selection of anew type of rabies vaccine and development of other disease vaccines based on thisnew system. This new system allows further study of pathogenic mechanism,selection of a new type of rabies vaccine as well development of RABV-basedvaccines for other disease.