Objective: To study the viral titers of live attenuated yellow fever vaccine in China under different storage conditions and time, and to provide data to support the stability of the vaccine. Methods: The viral titers of live attenuated yellow fever vaccines stored at different time points at -20 ℃, 4 ℃, 25 ℃ and 37 ℃ were determined; the viral titers of vaccines stored at different time points at the viral titers of vaccines expired for different time points were determined; the viral titers of vaccines were determined after storing at -20 ℃ after being taken away from the cold-chain during transportation; the titer of the virus was determined at different time points after thawing. Results: The viral titer of the live attenuated yellow fever vaccine stored at -20 ℃ for 24 months (validity period) decreased only by 0.4-0.5 LgPFU/ml; the viral titer decreased by 0.7 LgPFU/ml, 1.0-1.4 LgPFU/ml and 2.3-2.6 LgPFU/ml respectively when the vaccine was stored at 4 ℃, 25 ℃ and 37 ℃ for 8weeks. The viral titers decreased by 0.4-0.6 LgPFU/ml, 0.6-0.7 LgPFU/ml, 0.6-0.7 LgPFU/ml and 0.9 LgPFU/ml respectively 8 months 10 months, 44 months and 76 months after the expiration date. The titer of the virus decreased slightly from 0 to 0.2 LgPFU/ml when the vaccine was stored at 4 ℃ for 2 h, 25 ℃ for 2 h, 37 ℃ for 2 h, then -20 ℃ for 2 weeks. Afterthawing, the titers of virus decreased by 0.1-0.4 LgPFU/ml at room temperature for 120 minutes. Conclusions The live attenuated yellow fever vaccine in China has good stability and is very stable at the current standard storage temperature -20 ℃. Short time exposure to high temperature, whether in lyophilized state or after being thawed, the viral titer of vaccine remained almost unchanged.

Objective: To study the intracerebral pathogenicity of the yellow fever vaccine strains of Tiantan strain used in China and WHO vaccine strain 17D-213 in mice. Methods: Mice of different ages and strains were intracerebrally injected with same amount of Tiantan strain and 17D-213 strain. The death and survival of mice were observed and recorded. The LD₅₀/ml and half survival time of the two vaccine strains were compared and analyzed. Results: There was no difference in LD₅₀/ml between the Tiantan strain and 17D-213 strain when used through intracerebral injection in one-day-old suckling mice, 7-9 g mice or 12-14 g mice. Moreover, no significant difference in survival trend was found in 7-9 g mice or 12-14 g mice injected with the two vaccine strains. However, the two strains had statistically different influences on the survival trend of one-day-old suckling mice. The half survival time of the Tiantan strain was 11 d, while that of the WHO vaccine strain 17D-213 was 6 d. Excepting in NIH mice, no significant differences in LD₅₀/ml were detected between the same amount of two strains in BALB/c, KM, ICR or C57 mice. Conclusions The yellow fever Tiantan vaccine strain and WHO vaccine strain 17D-213 have no significant difference in the intracerebral pathogenicity in mice of different ages and strains with good safety.

Objective: To construct a stable infectious clone of live attenuated dengue virus (DENV) type 4 Ban18HK20 strain for better understanding the virulence determinants of DENV and improving the development of chimeric vaccines. Methods: Specific primers were constructed according to the genome of Ban18HK20 strain and used to subclone six cDNA fragments, which were linked into a high-copy plasmid pSPTM to obtain a stable full-length cDNA clone of DENV. RNA was transcribed from the full-length cDNA in vitro and electrotransfected into Vero cells to recover the virus. Biological characteristics of the recovered virus were identified using plaque assay, indirect immunofluorescence assay, growth kinetics test and pathogenicity study in mouse brain. Genetic stability of the virus passaged 15 generations was studied using RT-PCR amplification. Results: Restriction enzyme digestion and sequencing analysis indicated that the infectious clone was constructed successfully. The recovered virus was consistent with the parental virus in terms of plaque morphology, DENV E protein expression, growth characteristics and pathogenicity in mouse brain. Sequencing analysis showed that the recovered virus had the same genome sequence as the parental virus with good hereditary stability. Conclusions A stable infection clone of Ban18HK20 was constructed and a reverse genetics technology platform for DENV research was established.