Construction And Characterization Of A Missing Strain Of Rabies Virus With Shortened Intergenic Region Of CVS-11 Strain

Rabies is an od and widespread neurological zoometric disease which affects almost all kinds of mammals,including humans.The causal agent of the disease is rabies virus?RABV?,a number of lyssavirus genus,Rhabdoviridae family.The rabies virus genome is 11927nt in size consisting five monocistronic RNAs,which encodes the nucleocapsid protein?phosphoprotein?matrix protein?glycoprotein and large protein.Between the monocistronic there are four intergenic regions with different length,and the intergenic regions of five structural protein genes are 2?5?5?423nucleotides,respectively.The single-stranded negative-sense RNA genome structure is relatively simple.Therefore,it is beneficial to the operation of its genome.However,compared with vesicular stomatitis virus in the same family of Elastomatitis,the nucleotide sequence of the spacer region of RABV is unbalanced.How does the disequilibrium of the intergenic region of the RABV gene affect the virus in the end?It is a question worth thinking and studying.In this paper,the reverse genetic operating system of rabies virus was used to reconstruct the gene spacer region of rabies virus CVS-11 strain and to construct the missing strain with shortened interval region of rabies virus CVS-11strain.And further explore the effect of shortening the interval of the strain on the characteristics of each aspect of the CVS-11 strain.By sequencing the CVS-11 strain of rabies virus attenuated strain,the bases in the intergenic region of the gene were genetically modified so that the nucleotides in the intergenic region between the proteins were CT,and the whole length was amplified in four segments.Then the full-length cDNA clone vector of the virus was constructed by homologous recombination with in-fusion technique,two pairs of specific primers were amplified by PCR.The target bands of 1570 bp and 850 bp were obtained,respectively,which proved that homologous recombination was successful.Next,the co-transfection of the co-expression plasmids of N?P?L and G proteins with the full-length plasmids of the deleted rabies virus CVS-11 intergenic region was co-transfected into the BSR-T7 cell line,by saving recombinant virus,direct immunofluorescence detection and identification,and the specific fluorescence was observed under fluorescence microscope.It was proved that the missing strain of rabies virus CVS-11 strain with shortened intergenic region was successfully rescued and expressed in BSR-T7 cell line.The target band of 820bp was obtained by RT-PCR of the gene interval region of the G protein and L protein of the recombinant virus,and the deleted strain of the rabies virus CVS-11 interval region was confirmed to be stably transcribed in the cell.The typical characteristics of RABV particles can be observed under the transmission electron microscope by taking the cell culture of the recombinant virus and the original strain CVS-11.By drawing the recombinant virus and the original strain in different virus infection numbers,it was proved that the recombinant virus showed similar replication characteristics as the original strain,but the titer of the recombinant virus was lower than that of the original strain.Mice 10 mice in each group were infected with1×10⁷TCID₅₀/mL and1×10⁶TCID₅₀/mL recombinant virus and original strain respectively.After six days of infection,the incidence and death of the virus were recorded.The results showed that the deletion strain with shortened spacer of rabies virus CVS-11 strain had stronger lethal ability and lower survival rate,and the difference was significant.Mice?10 mice in each group?were intramuscularly inoculated with 1×10^7.5TCID₅₀/mL recombinant virus and protovirus strain with aluminum adjuvant.After 14 days of inoculation,blood was collected from eyeball,serum was isolated,and antibody was detected by fluorescent antibody neutralization test.The results showed that the shortening of the CVS-11 septum of rabies virus had no significant effect on it.In this study,we successfully constructed the missing strain of rabies virus CVS-11 strain,and studied its biological characteristics and immunogenicity.The results showed that the shortening of the septum of rabies virus CVS-11 strain had no significant effect on the replication and immunogenicity of rabies virus CVS-11strain,but it could enhance the pathogenicity of mice and the influence was obvious.