2株H7N9亚型重组禽流感病毒的生物学特性和免疫原性研究

The biological characteristics and immunogenicity of two ressortant H7N9 avian influenza viruses

为评估经反向遗传构建的H7N9亚型重组禽流感病毒(AIV)H7-Re1株和H7-Re2株的生物学特性及免疫原性,本研究将这2株病毒分别以10^4倍稀释接种10日龄SPF鸡胚后,36℃培养,观察鸡胚存活情况并检测病毒HA效价,结果显示96 h时,2株病毒接种的鸡胚全部存活,培养至72 h时其HA效价均为9 log2,均显著高于亲本分离株。测定2株病毒的静脉内接种致病指数(IVPI),结果显示均为0。将2株病毒分别以10^6 EID50剂量鼻腔内接种5周龄SPF鸡,进行致病性试验,结果显示,接种后14 d内,2株病毒接种鸡均无临床症状,也不排毒。以2株病毒为种毒分别制备油乳剂灭活疫苗,以0.3 mL/只接种3周龄SPF鸡后3周时检测免疫鸡血清中HI抗体,结果显示,两种疫苗免疫鸡平均HI效价均可达到8.0 log2。于免疫后21 d,H7-Re1株免疫组鸡分别攻击10^6 EID50的H7N9亚型低致病力AIV(PG/SHH/S1069/13株)和10^5 EID50的高致病力AIV(CK/GX/SD098/17株),进行攻毒试验,结果显示两种病毒攻毒后免疫鸡均全部存活,且无发病和排毒出现。H7-Re2株免疫组鸡于免疫后21 d分别以10^5 EID50的剂量进行高致病力H7N9亚型AIV(CK/GX/SD098/17株)和H7N2亚型AIV(DK/FJ/SE0195/18株)的攻毒保护试验,结果显示两种病毒攻毒后免疫鸡均不发病、不死亡、不排毒。以上结果表明,以H1N1亚型A/Puerto Rico/8/34株病毒为内部基因供体、经反向遗传学方法构建的H7-Re1株和H7-Re2株均具有生长滴度高、生物安全性高和免疫原性好的特点。本研究为H7N9亚型禽流感疫苗的研制和应用奠定了基础。

To evaluate the biological characteristics and immunogenicity of two ressortant H7N9 avian influenza viruses(H7-Re1 and H7-Re2)generated by reverse genetics,1:10 dilution of these two ressortant viruses were inoculated in 10-day-old chicken embryos,respectively,and cultured at 36℃for 96 hours.All the embryos remained alive during the culturation period and the HA titers of the two viruses in the allantoic fluid both reached up to 9 log2 at 72 hours post inoculation,which showed higher HA titers than those of wild-type viruses.The IVPI values of both two viruses were 0,and 5-week-old SPF chickens survived without any clinical signs or virus shedding after inoculation with 106 EID50 of the two viruses,respectively.One dose(0.3 mL)of inactivated oil-adjuvanted vaccine prepared from H7-Re1 or H7-Re2 ressortant viruses could induce high mean HI antibody titers of≥8 log2 to the vaccine antigen in SPF chickens at 3 weeks post vaccination.When challenged with 10^6 EID50 of the low pathogenic H7N9 virus(PG/SHH/S1069/13)or 10^5 EID50 of lethal H7N9 virus(CK/GX/SD098/17),all the chickens immunized with the H7-Re1 vaccine survived without any clinical signs or virus shedding.The chickens immunized with the vaccine prepared from H7-Re2 virus remained healthy without any virus shedding after challenge with 10^5 EID50 of the lethal H7N9(CK/GX/SD098/17)or H7N2(DK/FJ/SE0195/18)virus.The results showed that H7-Re1 and H7-Re2 ressortant viruses which bear the HA and NA genes from H7N9 viruses and the internal genes from A/Puerto Rico/8/34(H1N1/PR8)virus exhibited high yield in chicken embryos,good biosafety and sound immune efficacy.This study laid the foundation for the development and application of the H7 avian influenza vaccine in poultry.