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

Zhu S, Wang C, Zhang P, Li H, Luo S, Guo C - Virol. J. (2014)

Bottom Line: 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).Our results showed that CTNCEC25 was more closely related to wild street strains circulating in China than other vaccine strains.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.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Weiguang Biological Products Co,, Ltd, Shenzhen 518107, Guangdong province, China. gcpzxy0402@aliyun.com.

ABSTRACT

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.

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Analysis of sequence across the gene junctions of P and M genes. (A) Sequence analysis of the PCR product of the P and M gene junction in CTNCEC25. (B) Intergenic junction sequences of the P and M genes in 41 RABV genomes.
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Fig1: Analysis of sequence across the gene junctions of P and M genes. (A) Sequence analysis of the PCR product of the P and M gene junction in CTNCEC25. (B) Intergenic junction sequences of the P and M genes in 41 RABV genomes.

Mentions: The finding that a deletion of one A nucleotide was occurred in the gene junction of P and M was unexpected as several studies have shown that a 3’- U-U-U-U-U-U-U-5’ (U7) tract at the end of each gene was essential for the polyadenylated tail of the five structural protein genes and was well conserved in all these five structural protein genes [3, 33]. To further investigate the heterogeneity of the gene junctions of P and M, the CTNCEC25 P gene was used to searched against the nucleotide sequences at the NCBI database and a total of 41 RABV strains with complete genome sequenced was selected to compare the sequence heterogeneity of the P-M gene junctions. The summary of the sequence analysis is shown in Figure 1. The sequence across the P-M junction was wild type (with U7 tract) in 34 of 41 genomes and the shortening of U7 tract to U6 was found in three genomes including CTNCEC25. In the rest four genomes that were not wild type, the U tract was extended to U8 in three and interrupted by a C residue (U5CU1) in one genome (Figure 1). The above data showed that mutations that are known to eliminate termination of transcription might be selected and preserved in virus population during evolution.Figure 1


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

Zhu S, Wang C, Zhang P, Li H, Luo S, Guo C - Virol. J. (2014)

Analysis of sequence across the gene junctions of P and M genes. (A) Sequence analysis of the PCR product of the P and M gene junction in CTNCEC25. (B) Intergenic junction sequences of the P and M genes in 41 RABV genomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196109&req=5

Fig1: Analysis of sequence across the gene junctions of P and M genes. (A) Sequence analysis of the PCR product of the P and M gene junction in CTNCEC25. (B) Intergenic junction sequences of the P and M genes in 41 RABV genomes.
Mentions: The finding that a deletion of one A nucleotide was occurred in the gene junction of P and M was unexpected as several studies have shown that a 3’- U-U-U-U-U-U-U-5’ (U7) tract at the end of each gene was essential for the polyadenylated tail of the five structural protein genes and was well conserved in all these five structural protein genes [3, 33]. To further investigate the heterogeneity of the gene junctions of P and M, the CTNCEC25 P gene was used to searched against the nucleotide sequences at the NCBI database and a total of 41 RABV strains with complete genome sequenced was selected to compare the sequence heterogeneity of the P-M gene junctions. The summary of the sequence analysis is shown in Figure 1. The sequence across the P-M junction was wild type (with U7 tract) in 34 of 41 genomes and the shortening of U7 tract to U6 was found in three genomes including CTNCEC25. In the rest four genomes that were not wild type, the U tract was extended to U8 in three and interrupted by a C residue (U5CU1) in one genome (Figure 1). The above data showed that mutations that are known to eliminate termination of transcription might be selected and preserved in virus population during evolution.Figure 1

Bottom Line: 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).Our results showed that CTNCEC25 was more closely related to wild street strains circulating in China than other vaccine strains.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.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Weiguang Biological Products Co,, Ltd, Shenzhen 518107, Guangdong province, China. gcpzxy0402@aliyun.com.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus