Sequencing and molecular characterization of CTNCEC25, a China fixed rabies virus vaccine strain CTN-1 adapted to primary chicken embryo cells

Virology Journal

Caiping Guo¹  Shan Luo¹  Hui Li¹  Pei Zhang¹  Chunhua Wang¹  Shimao Zhu¹ 

[1] Shenzhen Weiguang Biological Products Co., Ltd, Shenzhen 518107, Guangdong province, China

关键词: Vaccine;    Chicken embryo cell;    CTNCEC25;    Rabies virus;    Genome sequence;   

Others  :  1148246
DOI  :  10.1186/1743-422X-11-176

 received in 2014-05-27, accepted in 2014-09-22,  发布年份 2014

【 摘 要 】

Background

Rabies virus is the main etiologic agent of the widespread neurological disease rabies. Recently, the China rabies virus vaccine strain CTN-1 adapted to chicken embryo cells, which has been designated as CTNCEC25, was obtained and demonstrated to have high immunogenicity. However, the full genome sequence of CTNCEC25 and its phylogenetic relationship with other rabies virus street and vaccine strains have not been characterized.

Results

The complete genome of CTNCEC25 was sequenced and analyzed. The length of CTNCEC25 genome is 11,924 nucleotides (nt), comprising a 3′ leader sequence of 59 nt, nucleoprotein (N) gene of 1,425 nt, phosphoprotein (P) gene of 989 nt, matrix protein (M) gene of 803 nt, glycoprotein (G) gene of 2,067 nt, RNA-dependent RNA polymerase gene (L) of 6,474 nt and a 5′ trailer region of 71 nt. A comparison of the entire genomes of CTN-1 and CTNCEC25 identified 16 nt substitutions and 1 deletion, resulting in 8 amino acid (aa) changes in the five structural proteins with one in L (aa 1602), two in M (aa 99 and 191) and six in mature G (aa 147, 333, 389, 421 and 485). The percentage homology of the CTNCEC25 genomic sequence with other fully sequenced rabies virus strains ranged from 81.4% to 99.9%. Phylogenetic analysis indicated that CTNCEC25 was more closely related with those recently isolated China street strains than other vaccine strains. Virus growth analysis showed that CTNCEC25 achieved high rate of propagation in cultured cells.

Conclusions

In this study, the complete genome of CTNCEC25 was sequenced and characterized. Our results showed that CTNCEC25 was more closely related to wild street strains circulating in China than other vaccine strains. Sequence analysis showed that the G protein ectodomain amino acid sequence identity between CTNCEC25 and other rabies virus strains was at least 90% identical. Furthermore, CTNCEC25 achieved high virus titers in cultured cells. Given that CTNCEC25 has high immunogenicity and induced strong protective immune response in animals, these results collectively demonstrated that CTNCEC25 is an ideal vaccine strain candidate for producing human vaccine with high quality and safety in China.

【 授权许可】

   
2014 Zhu et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Dietzschold B, Schnell M, Koprowski H: Pathogenesis of rabies. Curr Top Microbiol Immunol 2005, 292:45-56.
• [2]Zhang YZ, Xiong CL, Xiao DL, Jiang RJ, Wang ZX, Zhang LZ, Fu ZF: Human rabies in China. Emerg Infect Dis 2005, 11:1983-1984.
• [3]Tordo N, Poch O, Ermine A, Keith G, Rougeon F: Walking along the rabies genome: is the large G-L intergenic region a remnant gene? Proc Natl Acad Sci U S A 1986, 83:3914-3918.
• [4]Albertini AA, Ruigrok RW, Blondel D: Rabies virus transcription and replication. Adv Virus Res 2011, 79:1-22.
• [5]Mebatsion T, Weiland F, Conzelmann KK: Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 1999, 73:242-250.
• [6]Mebatsion T, Konig M, Conzelmann KK: Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 1996, 84:941-951.
• [7]Cox JH, Dietzschold B, Schneider LG: Rabies virus glycoprotein. II. Biological and serological characterization. Infect Immun 1977, 16:754-759.
• [8]Wiktor TJ, Gyorgy E, Schlumberger D, Sokol F, Koprowski H: Antigenic properties of rabies virus components. J Immunol 1973, 110:269-276.
• [9]Zhu JH, Wang J, Cai B, Zhao W, Zhu Y, Chao R, Chen L, Xue H, Ying BL, Li CP, Hu QL, Sha J, Esposito JJ: Immunogenicity and relative attenuation of different vaccinia-rabies virus recombinants. Arch Virol 1996, 141:1055-1065.
• [10]Faber M, Pulmanausahakul R, Hodawadekar SS, Spitsin S, McGettigan JP, Schnell MJ, Dietzschold B: Overexpression of the rabies virus glycoprotein results in enhancement of apoptosis and antiviral immune response. J Virol 2002, 76:3374-3381.
• [11]Dietzschold B, Wunner WH, Wiktor TJ, Lopes AD, Lafon M, Smith CL, Koprowski H: Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc Natl Acad Sci U S A 1983, 80:70-74.
• [12]Morimoto K, Hooper DC, Spitsin S, Koprowski H, Dietzschold B: Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol 1999, 73:510-518.
• [13]Morimoto K, Foley HD, McGettigan JP, Schnell MJ, Dietzschold B: Reinvestigation of the role of the rabies virus glycoprotein in viral pathogenesis using a reverse genetics approach. J Neurovirol 2000, 6:373-381.
• [14]Seif I, Coulon P, Rollin PE, Flamand A: Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J Virol 1985, 53:926-934.
• [15]Lentz TL, Wilson PT, Hawrot E, Speicher DW: Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins. Science 1984, 226:847-848.
• [16]Thoulouze MI, Lafage M, Schachner M, Hartmann U, Cremer H, Lafon M: The neural cell adhesion molecule is a receptor for rabies virus. J Virol 1998, 72:7181-7190.
• [17]Langevin C, Jaaro H, Bressanelli S, Fainzilber M, Tuffereau C: Rabies virus glycoprotein (RVG) is a trimeric ligand for the N-terminal cysteine-rich domain of the mammalian p75 neurotrophin receptor. J Biol Chem 2002, 277:37655-37662.
• [18]Tuffereau C, Benejean J, Blondel D, Kieffer B, Flamand A: Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus. EMBO J 1998, 17:7250-7259.
• [19]Benmansour A, Leblois H, Coulon P, Tuffereau C, Gaudin Y, Flamand A, Lafay F: Antigenicity of rabies virus glycoprotein. J Virol 1991, 65:4198-4203.
• [20]Dietzschold B, Gore M, Marchadier D, Niu HS, Bunschoten HM, Otvos L Jr, Wunner WH, Ertl HC, Osterhaus AD, Koprowski H: Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine. J Virol 1990, 64:3804-3809.
• [21]van der Heijden RW, Langedijk JP, Groen J, UytdeHaag FG, Meloen RH, Osterhaus AD: Structural and functional studies on a unique linear neutralizing antigenic site (G5) of the rabies virus glycoprotein. J Gen Virol 1993, 74(Pt 8):1539-1545.
• [22]Prehaud C, Coulon P, LaFay F, Thiers C, Flamand A: Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. J Virol 1988, 62:1-7.
• [23]Faber M, Faber ML, Papaneri A, Bette M, Weihe E, Dietzschold B, Schnell MJ: A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity. J Virol 2005, 79:14141-14148.
• [24]Takayama-Ito M, Ito N, Yamada K, Minamoto N, Sugiyama M: Region at amino acids 164 to 303 of the rabies virus glycoprotein plays an important role in pathogenicity for adult mice. J Neurovirol 2004, 10:131-135.
• [25]Takayama-Ito M, Ito N, Yamada K, Sugiyama M, Minamoto N: Multiple amino acids in the glycoprotein of rabies virus are responsible for pathogenicity in adult mice. Virus Res 2006, 115:169-175.
• [26]Rupprecht CE, Barrett J, Briggs D, Cliquet F, Fooks AR, Lumlertdacha B, Meslin FX, Muler T, Nel LH, Schneider C, Tordo N, Wandeler AI: Can rabies be eradicated? Dev Biol (Basel) 2008, 131:95-121.
• [27]Rupprecht CE, Hanlon CA, Slate D: Control and prevention of rabies in animals: paradigm shifts. Dev Biol (Basel) 2006, 125:103-111.
• [28]Du J, Zhang Q, Tang Q, Li H, Tao X, Morimoto K, Nadin-Davis SA, Liang G: Characterization of human rabies virus vaccine strain in China. Virus Res 2008, 135:260-266.
• [29]Jiao W, Yin X, Li Z, Lan X, Li X, Tian X, Li B, Yang B, Zhang Y, Liu J: Molecular characterization of China rabies virus vaccine strain. Virol J 2011, 8:521. BioMed Central Full Text
• [30]Cai L, Tao X, Liu Y, Zhang H, Gao L, Hu S, Liu F, Li H, Shen X, Liu J, Wang S, Tang Q: Molecular characteristics and phylogenetic analysis of N gene of human derived rabies virus. Biomed Environ Sci 2011, 24:431-437.
• [31]Badrane H, Bahloul C, Perrin P, Tordo N: Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity. J Virol 2001, 75:3268-3276.
• [32]Guo C, Wang C, Luo S, Zhu S, Li H, Liu Y, Zhou L, Zhang P, Zhang X, Ding Y, Huang W, Wu K, Zhang Y, Rong W, Tian H: The adaptation of a CTN-1 rabies virus strain to high-titered growth in chick embryo cells for vaccine development. Virol J 2014, 11:85-92. BioMed Central Full Text
• [33]Wertz GW, Moudy R, Ball LA: Adding genes to the RNA genome of vesicular stomatitis virus: positional effects on stability of expression. J Virol 2002, 76:7642-7650.
• [34]Prehaud C, Hellebrand E, Coudrier D, Volchkov VE, Volchkova VA, Feldmann H, Le Guenno B, Bouloy M: Recombinant Ebola virus nucleoprotein and glycoprotein (Gabon 94 strain) provide new tools for the detection of human infections. J Gen Virol 1998, 79(Pt 11):2565-2572.
• [35]Diallo A: Avirulent mutants of the rabies virus: change in site III of the glycoprotein. Ann Rech Vet 1986, 17:3-6.
• [36]Tuffereau C, Leblois H, Benejean J, Coulon P, Lafay F, Flamand A: Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 1989, 172:206-212.
• [37]Mebatsion T: Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein. J Virol 2001, 75:11496-11502.
• [38]Morimoto K, McGettigan JP, Foley HD, Hooper DC, Dietzschold B, Schnell MJ: Genetic engineering of live rabies vaccines. Vaccine 2001, 19:3543-3551.
• [39]Ito N, Takayama M, Yamada K, Sugiyama M, Minamoto N: Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice. J Virol 2001, 75:9121-9128.
• [40]Barr JN, Whelan SP, Wertz GW: cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation. J Virol 1997, 71:8718-8725.
• [41]Hwang LN, Englund N, Pattnaik AK: Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intercistronic gene junctions. J Virol 1998, 72:1805-1813.
• [42]Horton DL, McElhinney LM, Marston DA, Wood JL, Russell CA, Lewis N, Kuzmin IV, Fouchier RA, Osterhaus AD, Fooks AR, Smith DJ: Quantifying antigenic relationships among the lyssaviruses. J Virol 2010, 84:11841-11848.
• [43]Wu X, Smith TG, Rupprecht CE: From brain passage to cell adaptation: the road of human rabies vaccine development. Expert Rev Vaccines 2011, 10:1597-1608.
• [44]Manohar M, Orrison B, Peden K, Lewis AMJ: Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice. Biologicals 2008, 36:65-72.
• [45]Levenbook IS, Petricciani JC, Elisberg BL: Tumorigenicity of Vero cells. J Biol Stand 1984, 12:391-398.
• [46]Barth R, Gruschkau H, Bijok U, Hilfenhaus J, Hinz J, Milcke L, Moser H, Jaeger O, Ronneberger H, Weinmann E: A new inactivated tissue culture rabies vaccine for use in man. Evaluation of PCEC-vaccine by laboratory tests. J Biol Stand 1984, 12:29-46.
• [47]Kondo A: Pre-immunization and post-exposure treatment with inactivated rabies vaccine of chick embryo cell culture origin (CEC). Dev Biol Stand 1978, 40:147-153.
• [48]Nicholson KG, Farrow PR, Bijok U, Barth R: Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man. Vaccine 1987, 5:208-214.
• [49]Kamoltham T, Thinyounyong W, Phongchamnaphai P, Phraisuwan P, Khawplod P, Banzhoff A, Malerczyk C: Pre-Exposure Rabies Vaccination Using Purified Chick Embryo Cell Rabies Vaccine Intradermally is Immunogenic and Safe. J Pediatr 2007, 151:173-177.
• [50]Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23:2947-2948.
• [51]Smith JS, Yager PA, Baer GM: A rapid reproducible test for determining rabies neutralizing antibody. Bull World Health Organ 1973, 48:535-541.