摘要
为实现对H7N9禽流感病毒(AIV)感染过程的动态可视化示踪,本研究构建了重组报告质粒pHH21-NS-Venus、pHH21-NS-Apple 和包含 H7N9 AIV CK/SD008 株的 8 个基因节段的重组质粒 pHH21-PB2-627E、pHH21-PB2-627K(来自 CK/SD008-627K 株)、pHH21-PB1、pHH21-PA、pHH21-HA、pHH21-NA、pHH21-NP、pHH21-M。上述质粒均经测序鉴定。利用12质粒反向遗传操作系统,构建2株荧光报告病毒SD008-627EVenus、SD008-627E-Apple,及E627K 对哺乳动物嗜性的 2株荧光报告病毒 SD008-627K-Venus、SD008-627KApple。将这4株报告病毒感染A549细胞后在荧光显微镜下均可见相应荧光且病毒主要定位在细胞核中;将其中两株报告病毒SD008-627E-Venus/Apple经鸡胚传2代,2株SD008-627K-Venus/Apple在小鼠体内传3代后均感染A549细胞,经倒置荧光显微镜观察均可见相应荧光。上述结果表明4株报告病毒正确构建,且其可以在鸡胚或者小鼠体内传代(即SD008-627K-Venus/Apple为小鼠适应性报告病毒)。将4株报告病毒与亲本病毒分别感染C57BL/6小鼠,进行小鼠致病性试验。结果显示,报告病毒SD008-627E-Venus/Apple与其亲本病毒SD008-627E的MLD_(50)均>5 log_(10)EID_(50)/mL,且均对小鼠不致死;报告病毒SD008-627K-Venus/Apple与其亲本病毒SD008-627K的MLD_(50) 分别为1.6 log_(10)EID_(50)/mL、2.3 log_(10)EID_(50)/mL 和 1.8 log_(10)EID_(50)/mL;报告病毒 SD008-627E-Venus/Apple 与其亲本病毒SD008-627E感染小鼠的鼻甲、肺、脑、脾和肾的病毒滴度均<10^(2) EID_(50)/mL;SD008-627K-Venus/Apple及其亲本病毒SD008-627K的鼻甲和肺的病毒滴度均>10^(6) EID_(50)/mL,脑、脾和肾的病毒滴度均<102EID_(50)/mL。上述结果表明,构建的4株报告病毒均未改变亲本病毒对小鼠的致病性。利用噬斑试验,将报告病毒SD008-627K-Venus感染MDCK细胞,结果可见其能够引起细胞病变和表达荧光蛋白且荧光斑数量与噬斑数量相近。分别将报告病毒SD008-627K-Venus感染小鼠,3 d后取其肺脏研
In order to realize the dynamic visual tracing of H7 N9 avian influenza virus(AIV) infection process, the recombinant reporter plasmids were constructed in this study, including pHH21-NS-Venus and pHH21-NS-Apple and 8 gene segments recombinant plasmids containing p HH21-PB2-627 E, pHH21-PB2-627 K(from CK/SD008-627 K strain) pHH21-PB1, p HH21-PA,pHH21-HA, pHH21-NA, pHH21-NP, pHH21-M of H7 N9 AIV CK/SD008 strain. All the plasmids were identified by sequencing.Using the 12-plasmid reverse genetic operating system, two fluorescent reporter viruses SD008-627 E-Venus/SD008-627 E-Apple and two fluorescent reporter viruses SD008-627 K-Venus/SD008-627 K-Apple that are mammalian-tropic to E627 K were constructed.After A549 cells were infected with these four reporter viruses, the corresponding fluorescence could be visually detected under a fluorescence microscope and the viruses were mainly located in the nucleus. Two of the reporter viruses SD008-627 E-Venus/Apple,that were passaged two times in chicken embryos, and two strains SD008-627 K-Venus/Apple, that were passaged three times in mice, can infect A549 cells, and the corresponding fluorescence can be visually detected under the inverted fluorescence microscope. The above results showed that the four reporter viruses were constructed correctly and can be passaged stably in chicken embryos or mice(i.e. SD008-627 K-Venus/Apple is a mouse adaptive reporter virus). C57 BL/6 mice were infected with the four reporter viruses and the parental viruses to evaluate their pathogenicity. The results showed that the MLD_(50) of the reported virus SD008-627 E-Venus/Apple and its parent virus SD008-627 E were both >5 log_(10) EID_(50)/mL, and both were nonpatho-genic to mice;the MLD_(50) values of the reported virus SD008-627 K, SD008-627 K-Venus, SD008-627 K-Apple and its parent virus were 1.6 log_(10) EID_(50)/mL, 2.3 log_(10) EID_(50)/mL and 1.8 log_(10) EID_(50)/mL, respectively;the virus titeres in the turbinate, lung, brain, spleen and kidney of mice infected with the report
作者
于晓菲
万晓朋
李继清
姜永萍
YU Xiao-fei;WAN Xiao-peng;LI Ji-qing;JIANG Yong-ping(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第3期231-238,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31521005)
中央级公益性科研院所基本科研业务费专项(1610302017001)
