H5与H7亚型流感流行株嵌合病毒株的拯救及初步鉴定
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摘要
目的探讨H5与H7亚型流感流行株嵌合病毒的拯救并进行鉴定,为新型流感疫苗的研发奠定基础。方法采用脂质体转染的方法,首先依照A型流感病毒A/PR/8/34 HA嵌合片段的设计对H5 2.3.4.4谱系HA和H7亚型的HA进行改造,利用反向遗传技术拯救A/B型嵌合减毒株,对拯救成功的病毒株进行形态学和分子生物学鉴定,并测定病毒滴度;从中选择H5亚型嵌合减毒株以10~4EID_(50)/50μl的剂量滴鼻感染BALB/c小鼠,观察、记录小鼠的体重变化及死亡情况,并测定攻毒后第3 d及第6 d小鼠肺脏病毒复制能力。结果成功拯救出2种A/B型嵌合减毒株,分别命名为rA/B-H5-NS110和rA/B-H7-NS110,测序显示两株嵌合减毒株的序列与预期一致,并在电镜下观察到了流感病毒样粒子。rA/B-H5-NS110和rA/B-H7-NS110株在MDCK细胞上的病毒滴度均为10~(4.50)TCID_(50)/ml;在鸡胚上的病毒滴度rA/B-H5-NS110为10~(5.25)EID_(50)/ml,rA/B-H7-NS110株为10~(5.44)EID_(50)/ml。小鼠致病性实验中H5亚型嵌合病毒感染组小鼠与对照组相比体重无明显下降,攻毒后小鼠存活率100%;攻毒后第3 d可在小鼠肺脏内检测到嵌合减毒株rA/B-H5-NS110的轻微复制,第6 d时未检测到病毒复制。结论成功地拯救出2株包含有H5 2.3.4.4谱系HA基因以及H7N9流感病毒HA基因的嵌合减毒株,为B型流感病毒包装机制的研究以及新型流感疫苗的研发提供了新的思路。
Objectives To explore the rescue and identification of a chimeric virus of the H5 and H7 subtypes of influenza strains causing outbreaks and to lay the foundation for the development of new influenza vaccines. Methods Liposome transfection was used. First, HA of the 2.3.4.4 clade of the H5 subtype and H7 subtype was specifically altered according to the design of a chimeric fragment of HA of influenza A virus A/PR/8/34. Reverse genetics was used to rescue A/B chimeric attenuated strains. Rescued strains were identified based on morphology and molecular biology, and the viral titer was determined. The chimeric attenuated strain of the H5 subtype was used to infect BALB/c mice via nasal drip at a dose of 10~4 EID_(50)/50μl. Changes in body weight and the death of mice were observed and recorded, and the lungs were sampled and screened for the ability of the virus to replicate on day 3 and day 6 after challenge. Results Two types of A/B chimeric strains designated rA/B-H5-NS110 and rA/B-H7-NS110 were successfully rescued. Sequencing results indicated that the sequence of the two chimeric attenuated strains was consistent with expectations, and influenza virus-like particles were observed in electron microscopy. The viral titer of the rA/B-H5-NS110 and rA/B-H7-NS110 strains in MDCK cells was 10~(4.50)TCID_(50)/ml. The viral titer of the rA/B-H5-NS110 strain in chicken embryos was 10~(5.25) EID_(50)/ml. The viral titer of the rA/B-H7-NS110 strain in chicken embryos was 10~(5.44)EID_(50)/ml. The results of pathogenicity testing in mice indicated that there was no significant decrease in body weight of mice in the experimental group infected with the H5 subtype of the chimeric virus, compared to the control group. The survival rate of mice after challenge was 100%. Slight replication of chimeric attenuated strain rA/B-H5-NS110 was detected in the lungs of mice on day 3 after challenge. No titers were detected on day 6. Conclusion Two chimeric attenuated strains with H5 2.3.4.4 lineage HA and H7 N9 HA were successfully recused, providing a new avenue of thought for the study of the packaging mechanism of the influenza B virus and the development of a novel influenza vaccine.
Objectives To explore the rescue and identification of a chimeric virus of the H5 and H7 subtypes of influenza strains causing outbreaks and to lay the foundation for the development of new influenza vaccines. Methods Liposome transfection was used. First, HA of the 2.3.4.4 clade of the H5 subtype and H7 subtype was specifically altered according to the design of a chimeric fragment of HA of influenza A virus A/PR/8/34. Reverse genetics was used to rescue A/B chimeric attenuated strains. Rescued strains were identified based on morphology and molecular biology, and the viral titer was determined. The chimeric attenuated strain of the H5 subtype was used to infect BALB/c mice via nasal drip at a dose of 10~4 EID_(50)/50μl. Changes in body weight and the death of mice were observed and recorded, and the lungs were sampled and screened for the ability of the virus to replicate on day 3 and day 6 after challenge. Results Two types of A/B chimeric strains designated rA/B-H5-NS110 and rA/B-H7-NS110 were successfully rescued. Sequencing results indicated that the sequence of the two chimeric attenuated strains was consistent with expectations, and influenza virus-like particles were observed in electron microscopy. The viral titer of the rA/B-H5-NS110 and rA/B-H7-NS110 strains in MDCK cells was 10~(4.50)TCID_(50)/ml. The viral titer of the rA/B-H5-NS110 strain in chicken embryos was 10~(5.25) EID_(50)/ml. The viral titer of the rA/B-H7-NS110 strain in chicken embryos was 10~(5.44)EID_(50)/ml. The results of pathogenicity testing in mice indicated that there was no significant decrease in body weight of mice in the experimental group infected with the H5 subtype of the chimeric virus, compared to the control group. The survival rate of mice after challenge was 100%. Slight replication of chimeric attenuated strain rA/B-H5-NS110 was detected in the lungs of mice on day 3 after challenge. No titers were detected on day 6. Conclusion Two chimeric attenuated strains with H5 2.3.4.4 lineage HA and H7 N9 HA were successfully recused, providing a new avenue of thought for the study of the packaging mechanism of the influenza B virus and the development of a novel influenza vaccine.
引文
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[12] Alvarado-Facundo E,Gao Y,Ribas-Aparicio RM,et al.Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface[J].J Virol,2015,89(4):1975-85.
[13] Hsieh CF,Chen YL,Lin CF,et al.An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro[J].Sci Rep,2016(6):36015.
[14] Benton DJ,Wharton SA,Martin SR,et al.Role of neuraminidase in influenza A(H7N9) virus receptor binding[J].J Virol,2017,91(11):e02293-16.
[15] Guo H,Rabouw H,Slomp A,et al.Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces[J].PLoS Pathog,2018,14(8):e1007233.
[16] Byrd-Leotis L,Cummings RD,Steinhauer DA.The interplay between the host receptor and influenza virus hemagglutinin and neuraminidase[J].Int J Mol Sci,2017,18(7):1541.
[2] He J,Duan J.First human case of avian influenza A (H5N6) in Yunnan province,China[J].SAGE Open Med Case Rep,2015(3):2050313X15596484.
[3] Yu Z,Gao X,Wang T,et al.Fatal H5N6 avian influenza virus infection in a domestic cat and wild birds in China[J].Sci Rep,2015(5):10704.
[4] Yang L,Zhu W,Li X,et al.Genesis and spread of newly emerged highly pathogenic H7N9 avian viruses in mainland China[J].J Virol,2017,91(23):e01277-17.
[5] Ke C,Mok CKP,Zhu W,et al.Human infection with highly pathogenic avian influenza A(H7N9) virus,China[J].Emerg Infect Dis,2017,23(8):1332-40.
[6] Baker SF,Nogales A,Finch C,et al.Influenza A and B virus intertypic reassortment through compatible viral packaging signals[J].J Virol,2014,88(18):10778-91.
[7] Hai R,Garcia-Sastre A,Swayne DE,et al.A reassortment-incompetent live attenuated influenza virus vaccine for protection against pandemic virus strains[J].J Virol,2011,85(14):6832-43.
[8] Hai R,Martinez-Sobrido L,Fraser KA,et al.Influenza B virus NS1-truncated mutants:live-attenuated vaccine approach[J].J Virol,2008,82(21):10580-90.
[9] 田明明,魏雪玲,杨兴,等.H5N8流感病毒密码子偏爱性及聚类分析[J].中国病原生物学杂志,2018,13(5):494-8,503.
[10] Horimoto T,Takada A,Iwatsuki-Horimoto K,et al.Generation of influenza A viruses with chimeric (type A/B) hemagglutinins[J].J Virol,2003,77(14):8031-8.
[11] Skehel JJ,Wiley DC.Receptor binding and membrane fusion in virus entry:the influenza hemagglutinin[J].Annu Rev Biochem,2000(69):531-69.
[12] Alvarado-Facundo E,Gao Y,Ribas-Aparicio RM,et al.Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface[J].J Virol,2015,89(4):1975-85.
[13] Hsieh CF,Chen YL,Lin CF,et al.An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro[J].Sci Rep,2016(6):36015.
[14] Benton DJ,Wharton SA,Martin SR,et al.Role of neuraminidase in influenza A(H7N9) virus receptor binding[J].J Virol,2017,91(11):e02293-16.
[15] Guo H,Rabouw H,Slomp A,et al.Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces[J].PLoS Pathog,2018,14(8):e1007233.
[16] Byrd-Leotis L,Cummings RD,Steinhauer DA.The interplay between the host receptor and influenza virus hemagglutinin and neuraminidase[J].Int J Mol Sci,2017,18(7):1541.
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