B型流感病毒B/Yamagata/16/88反向遗传操作平台的搭建及BALB/c小鼠感染模型的建立

目的 利用基因工程技术全基因合成B型流感病毒B/Yamagata/16/88的8个基因节段,并利用反向遗传技术从体外拯救B型流感病毒B/Yamagata/16/88,同时建立BALB/c小鼠感染模型,为下一步研究B型流感病毒致病机制、传播机制以及开发新型疫苗奠定基础。方法 通过基因合成和反向遗传技术体外拯救B型流感病毒B/Yamagata/16/88。全基因组测序验证拯救病毒基因组序列与Genbank序列的一致性。将拯救病毒以10⁵EID₅₀的攻毒剂量人工感染BALB/c小鼠,通过体重变化、生存率、肺脏病毒复制等方面进行致病性分析,建立小鼠感染模型。结果 成功从体外拯救出B型流感病毒B/Yamagata/16/88,命名为B-S9。全基因组测序结果表明,B-S9基因组序列与Genbank公布序列一致。B-S9能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠呈现低致病性;攻毒后第3天,B-S9感染小鼠体重出现下降,攻毒后第8天,小鼠体重开始回升;攻毒后第3天和第6天,B-S9感染小鼠的肺脏内均能检测到病毒复制,且攻毒后第3天的小鼠肺脏病毒滴度比攻毒后第6天的小鼠肺脏滴度高132倍。结论 成功搭建B型流感病毒B/Yamagata/16/88反向遗传操作平台,并建立BALB/c小鼠感染模型。目前国内外对B型流感病毒的研究比较少,该反向遗传操作平台的建立为B型流感病毒致病机制和传播机制的研究奠定了基础,同时也为包括B型流感病毒减毒活疫苗在内的新型疫苗的研制开辟了新途径。

Establishment of a reverse genetics system of influenza B virus B/Yamagata/16/88 and a BALB/c mouse model of influenza B virus infection

Objective To establish a BALB/c mouse model of influenza B virus infection, and to provide a foundation for studying the pathogenesis, mechanism of transmission of influenza B virus infection and developing new vaccines against influenza B virus. Methods Using genetic synthesis and reverse genetic technology, influenza B virus was rescued in vitro. We used the whole genome sequencing approach to validate the identity between the rescued viral genome sequences and the sequences reported in Genbank. To establish the BALB/c mouse model of influenza B virus infection, BALB/c mice were infected with 10⁵ EID₅₀ dose of the rescued virus, and the weight change, survival rate, and viral replication in the lungs were analyzed. Results We successfully rescued influenza B virus B/Yamagata/16/88 in vitro, and this virus was named B-S9. The genome sequencing results showed that the genome sequences of B-S9 was consistent with the GenBank-reported sequences. BALB/c mice were artificially infected with B-S9, and no death due to infection was observed. The above results indicated that B-S9 is of low pathogenicity to the BALB/c mice. The mice infected with B-S9 showed body weight decline in 3 days post inoculation (dpi) but restored in 8 dpi. The virus titers could be detected in the lungs of mice infected with B-S9 on dpi 3 and dpi 6, respectively. Furthermore, the virus titer in the mouse lungs on dpi 3 was 132 times higher than that on dpi 6. Conclusions A reverse genetic system of influenza B virus B/Yamagata/16/88 is successfully established, and a BALB/c mouse model of influenza B virus infection is established. To date, studies of influenza B virus are limited at home and abroad. The establishment of this reverse genetic system provides not only a platform for studying the pathogenesis and mechanism of transmission of influenza B virus, but also provides a way for developing new live-attenuated influenza B virus vaccine.