Construction Of A Recombinant Coxsackie Virus CA16-A6VP And Study On Its Infectivity

Coxsackievirus A16(CV-A6)is one of the more frequent pathogens causing hand,foot,and mouth disease(HFMD)in recent years.Current studies have shown that coxsackievirus group A can be cultured in human malignant embryonic rhabdomyoblastoma cells(RD),but it is difficult to culture in commonly used vaccine-producing stromal cells(such as Vero,MRC-5,etc.).The development of Coxsackie virus A group 6 virus vaccine has brought great obstacles.At present,there is a lack of specific drugs and vaccines for CV-A6 treatment,so the mechanism of cell infection of CV-A6 is studied,and related.The development of monovalent multivalent vaccines is imminent.CV-A6 does not undergo intermediate stages of DNA during replication,making recombinant DNA technology not directly applicable to the analysis and research of viral genomes,and the in vitro instability of viral RNA molecules has brought great difficulties to research.There is an urgent need for a genetic manipulation technique for RNA viruses.After several decades of development,reverse genetics technology has solved this problem by obtaining the complete c DNA of the viral genome through genetic manipulation technology,and then preparing the resulting c DNA into an infectious transcript.This infectious transcript can The virions are retrieved in the host cell.This technique,which allows the RNA virus to undergo an intermediate stage of DNA under artificial conditions,helps us to conduct an in-depth study of the CV-A6 virus at the DNA level,and we also operate CV-A6 through relevant genetic levels.The genome was engineered to explore the mechanism of CV-A6-associated cell infectivity,and an attempt was made to construct a recombinant virus with both CV-A6 and CV-A16 antigenic sites for subsequent development of Coxsack CV-A6 and CV-The A16 bivalent vaccine provides the basis.In the first part,we first extracted and isolated the CV-A6 virus in the herpes-like specimens of patients with hand,foot and mouth disease,and obtained seven strains of virus by plaque purification,and studied the cell infectivity of seven strains.We found that seven strains of virus can successfully infect human malignant embryonic rhabdomyosarcoma cells(RD).However,these seven strains are unable to infect Vero cells,which also poses a significant obstacle to the development of CV-A6 vaccine.CV-A16,another member of the hand-foot-and-mouth disease Coxsackie virus,can simultaneously infect RD and Vero cells,which has aroused our interest and attention.Finally,we identified the CV-A6 strain,and identified the gene sequences of seven strains of CV-A6 by electrophoresis and sequencing.Based on this,the phylogenetic tree map was analyzed by software and the toxicity was determined.The titer of the strain was selected and the highest titer CV-A6 strain XS45 was selected for subsequent experiments.In the second part,we constructed the full-length c DNAs of CV-A6 and CV-A16 using reverse genetics.Based on this study,the recombinant virus(CA16-C16VP)genomic c DNA was spliced by Overlap PCR.The recombinant virus(CA16-C16VP)c DNA sequence was replaced by the CA16-VP segment and inserted into the upstream and downstream CA6-VP segments.A suitable cleavage site was obtained with the T7 promoter sequence.By linking the c DNAs of the above three viruses to the p BM16A-TOPO vector,we constructed three p BM16A-CA6,p BM16A-CA16,p BM16A-Re(CA16-A6VP)infectious clones.Identification by sequencing,consistent with the theoretical sequence of the design,laid the foundation for our third part identification and its infective exploration.In the third part,we will explore the cell infectivity studies of the three infectious clones of p BM16A-CA6,p BM16A-CA16,and p BM16A-Re(CA16-A6VP).We linearized the three infectious clones by designing the restriction enzyme sites,transfected into RNA in vitro,and then co-transfected the cells by liposome,blindly passed three generations of adaptation,and launched an infection-related study on recombinant virus..We analyzed the genome sequences of three blind-transformed recombinant viruses,virus indirect immunofluorescence,cell microscopic morphology observation,and titer detection.We found that the recombinant CV-A6 constructed by reverse genetics,CV-A16 has similar traits to the parental strain,and is basically consistent in cell infectivity.The recombinant virus(CA16-A6VP)constructed was based on CV-A16,and CV-A6-VP was replaced by its VP segment.In the subsequent titer determination,the titer of the recombinant virus is higher than that of the parental viruses CV-A16 and CV-A6,but the cell infectivity is consistent with the infectivity of the CV-A6 virus,that is,it can infect the RD cells.Vero cells cannot be infected normally.This phenomenon of cell infection suggests that the structure of the VP segment of the virus may be an important structural basis for the relevant sites of virus-infected cells.There may be related gene regulatory proteins in the VP segment of the virus,which may affect the pathway of virus adsorption.Experimental Results:1.Seven strains(A: XS45,B: JB66,C: JB70,D: IB69,E: JB71,F: XS46,G: JB68)were successfully isolated from herpes samples of patients and studied for cell infectivity.2.In this study,CV-A6 and CV-A16 infectious clones were constructed using reverse genetic technology and transfected into cells.After whole-gene sequencing analysis,rt-pcr and immunofluorescence identification,the recombinant viruses cv-a6 and cv-a16 were successfully obtained.3.CV-A6,CV-A16 recombinant viruses showed the same cellular infectivity as the mother wild strains.4.On this basis,the virulence of the recombinant virus(CA16-A6VP)constructed was significantly higher than that of the original parents.The recombinant virus(CA16-A6VP)showed the same cellular infectivity as the cv-a6 maternal virus,which infected RD cells and could not be cultured on Vero cells.5.The cell infectious infection of cv-a6 should be related to its viral VP structure,where relevant regions(P1,P2,P3)sites may regulate its cell infectious ability.6.A stable set of upstream molecular biology technology has been established,through which relevant structures and sites can be replaced and mutated,and relevant molecular biology research on coxsackievirus and bivalent and polyvalent chimeric vaccines can be carried out.In summary,using reverse genetics,we can artificially construct recombinant viruses suitable for research.We can try to study viral gene-related functions by designing sites and targeting mutations,or replacing whole genes.Studying the pathogenic mechanism of virus-infected cells provides a good platform and material for the development of multivalent vaccines,laying the foundation for the subsequent research and development of vaccines and other aspects.