ABSTRACT
Objective@#To investigate the genetic stability of virus seed H2M20K7 (K7) of live attenuated Hepatitis A virus H2 strain (HAV, H2 strain) for production of hepatitis A (Live) vaccine, lyophilized after continuous passages.@*Methods@#The virus seed K7 of H2 strain was proliferated and passaged in KMB17 cells in cell factories. Viruses of different passages were harvested after continuous passages. Virus RNA was extracted and the complete genomes of different virus passages (K7, K10, K11, K13, K15, K18) were sequenced by using next-generation deep sequencing. The mutation rates of different passages were compared. The infectivity titers of different virus passages of H2 strain were tested by ELISA.@*Results@#The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed. The structure of gene was stable and non-synonymous mutation rate was lower than 0.57%. The mutation rate of 5 ’non-coding regions was lower than 0.1%. There was no significant mutation in VP1/2 A and 2C virulence site. The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID50/ml. No statistically significant difference was found in this study.@*Conclusions@#The gene structure of the master virus seed, working seed and different passages of H2M20K7 after subculture was stable and the mutation rate was low. No significant mutation was found in 5’non-coding regions, and the critical virulence sites such as VP1/2 A, 2B and 2C showed attenuated characteristics with low mutation rate. Virulence of the virus did not changed. The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.
ABSTRACT
<p><b>OBJECTIVE</b>To characterize the complete genome sequence of coxsackievirus B5 (CVB5)A210/KM/09 strain which was isolated from Yunnan, China, 2009.</p><p><b>METHODS</b>Eight overlapping clones covering the whole viral genome (excluding the poly-A tail)were obtained by RT-PCR and sequenced, with their nucleotide and amino acid sequences compared with other known CVB5 strains.</p><p><b>RESULTS</b>The genome of the CVB5 A210/KM/09 strain had 7 372 nucleotides in length, and containing a 742-nt non-translated region (NTR) at the 5' end and a 98-nt NTR at the 3' end. The entire open reading frame contained 6 555 nt, encoding a 2 185-aa polyprotein. In the coding region, there appeared no nucleotide deletion or insertion, but some changes of amino acid seemed unique. Based on the complete genome sequence alignments, CVB5 isolate A210/KM/09 strain showed the highest nucleotide (92.5%) and amino acid (97.3%) identities to the CVB5/CC10/10. It also shared nucleotide (80.1%-92.5%) and amino acid (95.0%-97.3%) homology with other CVB5 strains: 17Y, 19CSF, 20CSF, 1954/85/US, 2000/CSF/KOR, 03001N, CoxB5/Henan/2010, VB5/SD/09 and Faulkner. Blast between genome fragments, A210/KM/09 showed similarity on nucleotide (80.1%-92.5%) and amino acid (95.0%-97.3%) identities with other CVB5 strains. The phylogenetic tree, constructed on the complete VP1 regions, indicated that CVB5 could be divided into genotype A, B, C and D. while Genotype C and D could be further divided into C1-C4 and D1-D4 subgenotypes.</p><p><b>CONCLUSION</b>A210/KM/09 and other CVB5 predominant strains isolated in China belonged to CVB5 subgenotype C4.</p>