Study On The Attenuation And Anti-tumor Effect Of Coxsackievirus B Type

Objective Screening of CV-B5 attenuated strain by low-temperature passaging,verification of virulence loci by reverse genetics,and obtaining a CV-B5 attenuated strain that maintains oncolytic ability of the original strain CV-B5/Faulkner,providing a basis for further development of CV-B5 oncolytic therapy.Method CV-B5/Faulkner was passaged at low temperature（33℃）to the 40th passage,and the virulence and oncolysis of CV-B5/Faulkner,the 30th and 40th passages were compared.Than sequence the 10th,20th,30th and 40th generation strains.To identify the key attenuating sites of attenuated CV-B5,a total of 7 mutant strains of single site and combined site were obtained by reverse genetics method.The biological characterization of each mutant strain was studied by methods such as cytopathic effect（CPE）,growth kinetics,virus titer（CCID50）and challenge of suckling mice.To further studying the viral load and pathological changes in different tissues and organs of suckling mice.Comparison of the oncolytic capacity of the N725K mutant strain and CV-B5/Faulkner against lung and colon cancer cell lines.Using murine lung cancer cells and colon cancer cells with coxsackievirus-adenovirus receptor（CAR）to verify whether the N725K mutant strain can infect cells by CAR.In addition,the immunogenicity and challenge protection effects of CV-B5 mutant attenuated strains were investigated.Results The results of the challenge of suckling mice showed that the virulence of the30th and 40th generation viruses obtained by low temperature passage was significantly lower than that of CV-B5/Faulkner（P<0.0001,P<0.001）,but the oncolytic effect on NCI-H1299 lung cancer was Similar.The results of whole genome sequencing showed that there were 3 stable amino acid change sites since the 20th generation,namely G4L,H650Y,and N725K.Seven mutant strains were obtained by reverse genetics,namely G4L,H650Y,N725K,G4L+H650Y,G4L+N725K,H650Y+N725K,G4L+H650K+N725K.The CPE,growth kinetics and CCID₅₀ of the 7 mutants were similar to CV-B5/Faulkner.The results of the single-site mutant strain of suckling mice challenge experiment showed that the LD₅₀ of the G4L,H650Y and N725K mutants were 3.372×10⁶,1.896×10⁵,>1.896×10⁷ CCID₅₀(the original strain LD₅₀ was 5.996×10⁵ CCID₅₀),indicating that The virulence of both G4L and N725K mutants was significantly reduced,and the virulence of N725K was lower than that of G4L.The results of combined site mutant strain challenge showed that the LD₅₀of G4L+N725K,H650Y+N725K,G4L+H650K+N725Kmutantstrainswere5.996×10⁶,>1.896×10⁷,>1.896×10⁷ CCID₅₀,respectively,which also indicated that N725K was key attenuating sites for CV-B5/Faulkner.The viral loads in the pancreas,brain,spine and hind legs of the N725K mutant challenge group were significantly lower than those in the CV-B5/Faulkner group 4 days after infection.The degree of lesions in the brain and spine and the positive immunohistochemical reaction were lower than those in the CV-B5/Faulkner group,indicating that the neuropathogenicity of the N725K attenuated strain was weaker.The oncolytic ability of N725K attenuated strain against lung cancer cell lines（NCI-H1299,A549,NCI-H460）and colon cancer cell lines（SW620,SW480,HCT116,Lo Vo）was not significantly different from that of CV-B5/Faulkner,and all were dose-dependent.The N725K attenuated strain and CV-B5/Faulkner do not have the ability to infect mouse Lewis lung cancer（LLC）and mouse colon cancer cells CT26.WT that do not express CAR,while the above two cell lines stably transfected with CAR（LLC-CAR,CT26WT-CAR）were both sensitive to the N725K attenuated strain and CV-B5/Faulkner,suggesting that the two strains rely on the same receptors to infect cells.The results of in vivo anti-tumor experiments suggest that the N725K attenuated strain and CV-B5/Faulkner have consistent therapeutic effects in mice with transplanted NCI-H1299,LLC-CAR,or CT26.WT-CAR cell.One-shot immunization of mice with the attenuated N725K strain induced higher levels of neutralizing antibodies and had cross-neutralizing ability against CV-B5/Faulkner and CV-B5/JS417（GⅠ.D3 subtype）.After booster immunization,the neutralizing antibodies of N725K attenuated strain,CV-B5/Faulkner and CV-B5/JS417 strains increased by 16.1,7.0 and 11.7 times,respectively.The immune serum of N725K attenuated strain could effectively protect the lethal doses of CV-B5/Faulkner and CV-B5/JS417 against the challenge of suckling mice,with ED₅₀of 1:3.7 and 1:256.1,respectively.Maternal antibodies produced by one or two doses of attenuated N725K attenuated strains could effectively protect suckling mice against lethal doses of CV-B5/Faulkner and CV-B5/JS417.Conclusion In this study,an attenuated CV-B5 strain that maintained oncolytic effect was obtained by low-temperature passage.The reverse genetics method was used to determine the two virulence loci of CV-B5/Faulkner,which are G4L and N725K,It was found that the N725K attenuated strain had a good oncolytic effect on lung cancer and colon cancer,which provided a basis for CV-B5 to be used for oncolytic therapy.