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.

Biliary atresia is a rare infant disease that predisposes patients to liver transplantation and death if not treated in time. However, early diagnosis is challenging because the clinical manifestations and laboratory tests of biliary atresia overlap with other cholestatic diseases. Therefore, it is very important to develop a simple, safe and reliable method for the early diagnosis of biliary atresia. Herein, a novel NIR-II fluorescence probe, HZL2, with high quantum yield, excellent biocompatibility, low cytotoxicity and rapid excretion through the liver and gallbladder was developed based on the oil/water partition coefficient and permeability. A simple fecal sample after injection of HZL2 can be used to efficiently identify the success of the mouse model of biliary atresia for the first time, allowing for an early diagnosis of the disease. This study not only developed a simple and safe method for the early diagnosis of biliary atresia with great potential in clinical translation but also provides a research tool for the development of pathogenesis and therapeutic medicines for biliary atresia.