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

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

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。

Objective To establish a BALB/c mouse model of influenza B virus infection, and to provide a foun- dation 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 res- cued in vitro. We used the whole genome sequencing approach to validate the identity between the rescued viral genome se- quences and the sequences reported in Genbank. To establish the BALB/c mouse model of influenza B virus infection, BALB/e mice were infected with 105 EIDs0dose of the rescued virus, and the weight change, survival rate, and viral repli- cation 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 ob- served. 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 in- fluenza B virus are limited at home and abroad. The establishment of this reverse genetic system provides not only a plat- form for studying the pathogenesis and mechanism of transmission of influenza B virus, but also provides a way for develo- ping new live-attenuated influenza B virus vaccine.