| Classical swine fever?CSF?is very dangerous for pig industry and its prevention and control measures are mainly based on vaccine injection.hog cholera lapinized virus?HCLV?,which has made significant contribution to the prevention and control of CSF in China and even the world,is recognized as the gold standard attenuated vaccine in the world.The antibodies produced by CSF live vaccine immunized herds and wild-type Classical swine fever virus?CSFV?infection cannot be distinguished.In recent years,there has been a new genetic subtype CSFV in CSF live vaccine immunized herds,Which is a new challenge for the prevention and control of CSF in our country.Therefore,it is extremely important to understand the genetic evolutionary relationship of CSFV and to develop a novel CSF-marker vaccine that can differentiate infectious and vaccinated animals?DIVA?.In order to understand the genetic evolution of the CSFV E2 gene,this study collected20 suspected CSF diseases and amplified and sequenced partial genes of E2 by RT-PCR method.Three CSFV isolates were obtained and named GDFS1-2017-China,GDFS2-2017-China and GDQY-2018-China.E2 partial gene sequences?190 bp?were compared with the registered Chinese CSFV reference strains in GenBank,and the genetic evolution relationship was analyzed.The results showed the nucleotide homology with the CSF vaccine strain was 78.9%to 81.6%,and the nucleotide homology with the CSF Shimen strain was 78.9%to 82.1%.Phylogenetic relationship analysis showed that all 3CSFV isolates belonged to 2.1c gene subgroup.In order to further explore the relationship between the population evolution rate of CSFV strain,the beast v1.8.3 of the Bayesian Markov Chain Monte Carlo method was used to analyze the CSFV genes of different regions and different times registered in GenBank.The average nucleotide substitution rate is 3.1045×10-44 nucleotides/sites/year(95%HPD:3.0×10-4,3.3184×10-4).The CSFV phylogenetic tree shows that the nearest common ancestor?TMRCA?of the CSFV strain originated in 1489,which is more than 500 years ago.CSFV produced two major evolutionary branches in the middle of the fifteenth Century and in the early seventeenth Century,and most of the CSFV strains found since the 90 s last century came from the evolutionary branches of the early seventeenth Century.The results of CSFV group dynamic analysis showed that the outbreak of CSFV was generally on the rise,and the trend was relatively slow before eighteenth Century.It experienced two rapid growth in the early eighteenth Century and the early nineteenth Century,and the virus population declined rapidly in the late twentieth Century.In this study,the full-length HCLV gene was synthesized using the HCLV gene sequence?GenBank accession number:AF531433.1?as a reference.In order to obtain a precise CSFV genome RNA,we inserted the CMV promoter containing enhancers,Intron,T7 promoter,and Hammer ribozyme?HamRz?sequences at the 5'-UTR end of the whole genome sequence.And the 3'-UTR end was inserted into the Hepatitis delta virus ribozyme?HdvRz?,T7 terminator and SV40 PolyA sequences.At the same time,the marker gene was inserted at the end of the HCLV E1 gene,then the recombinant plasmid was constructed.A HCLV reverse genetic operation system based on RNA polymerase II was established,which directly transfected the full-length infectious clones with CMV as promoter and transfected the pig source susceptible cells to rescue the virus.The recombinant plasmid CN5266-1-PSK was transfected into SK-6 and BHK-21 cells,and the blank cells were set as the control group.The transfected cells were passed on 3 generations.Every generation was tested for virus RNA by RT-PCR and IFA detection with monoclonal antibodies to CSFV E2.The results showed that PCR did not amplify the specific target bands,and the specific fluorescence of vC-FLAG was not found in SK-6 cells,indicating that there was no virus particles to save vC-FLAG.On the basis of recombinant plasmid CN5266-1-PSK,in order to construct the infected clone of the labeled virus strain successfully,we verify our virus rescue system from two aspects of the labelled virus itself and the intracellular rescue system.First,the recombinant plasmid CN5266-1-mut?cut flag?-SwaI-PSK?vaccine strain vC?and CN5266-1-mut-SwaI-PSK?marker strain vC-FLAG?were established.Then the two recombinant plasmids were linearized in vitro,and the RNA which was obtained by transcription was transfected to SK-6 cells by Lipfectamine.The transfected cells were blindly transmitted for 3 generations,and each generation was tested for IFA detection with monoclonal antibodies to CSFV E2.The results showed that the specific fluorescence of the strains vC and vC-FLAG was not seen in the SK-6 cells,indicating that no rescue was performed.Viruses of strains vC and vC-FLAG.The transfected cells were blinded to 3 generations,and each generation was detected by CSFV E2 monoclonal antibody for IFA.The results showed that the specific fluorescence of vC and vC-FLAG was not found in SK-6 cells,indicating that there was no virus particles to save the vC and vC-FLAG of the virus.In order to verify the function of the main functional original CMV promoter,HamRz and HdvRz in the intracellular reverse genetic system.The recombinant plasmid CN5266-1-EGFP were constructed and transfected into SK-6 cells.The fluorescent cells can be seen obviously under the fluorescence microscope after 48 h,indicating that CMV promoter plays a role in SK-6 cells.Then the recombinant plasmid CN5266-1-EGFP?include HamRz functional site?and CN5266-1-EGFP?cut GTC??remove HamRz functional site?were constructed and transfected into SK-6 cells.The total protein of the transfected cells was extracted and the Western blot was detected after 48 h.The results showed that the expression of the gene expression in CN5266-1-EGFP?cut GTC?was obviously higher than that of the SK-6 cells,showingthatHamRzcanfunction.Finally,therecombinantplasmid CN5266-1-EGFP-FLAG and CN5266-1-EGFP-FLAG?cut HdvRz?were constructed on the basis of recombinant plasmid CN5266-1-EGFP?removing HdvRz sequence?and transfected into SK-6 cells.The total protein of the transfected cells was extracted and the Western blot was detected after 48 h.The results showed that there were two forms of EGFP in recombinant plasmid CN5266-1-EGFP-FLAG,but there was no significant difference in the expression of FLAG gene in CN5266-1-EGFP-FLAG?cut HdvRz?and CN5266-1-EGFP-FLAG,indicating that HdvRz could play a cutting effect,but the cutting efficiency was not high.In summary,the genetic evolution of the E2 gene of CSFV was analyzed in order to provide a theoretical basis for the screening of vaccine candidate strains from current epidemic strains.At the same time,a reverse genetic system for the attenuated strains of swine fever was constructed,and the different functional components in the reverse genetic system were verified,which lay the foundation for the development of the next CSF labelled vaccine. |
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