采用反向遗传学技术构建H5亚型禽流感疫苗株

目的构建重组H5亚型禽流感疫苗株。方法采用RT-PCR技术，分别扩增鹅源高产禽流感病毒A／Goose／Dalian／3／01（H9N2）的6条内部基因片段、高致病性禽流感病毒株A／Goose／HLJ／QFY／04（H5N1）的血凝素（HA）基因和N3亚型参考株A／Duck／Germany／1215／73（H2N3）的神经氨酸酶（NA）基因，并对HA1和HA2连接肽处的5个碱性氨基酸（R-R-R-K-K）的编码序列进行缺失与修饰，然后分别构建这8个基因的转录与表达载体，将其共转染293T／MDCK混合培养细胞单层，对拯救出的重组病毒进行表型分析。结果利用反向遗传学技术拯救出了全部基因都源于禽流感病毒的疫苗株，其基因序列符合设计要求包括删除HA基因的毒力相关序列，疫苗株的表型为H5N3。结论构建成功重组禽流感疫苗株rH5N3，为制备H5亚型禽流感疫苗打下了坚实的基础。     

H5N1流感疫苗株在Vero细胞中的研究[英文]

目的产生在Vero(非洲绿猴肾)细胞中能高适应生长的重配H5N1流感病毒疫苗株,并对其生物学特性进行初步测定。方法修饰A/Puerto Rico/8/34(H1N1)(PR8)的NS基因为Vero细胞适应型,并合成NS基因片段,构建质粒pHW2000-NS;通过RT-PCR方法,扩增疫苗株A/Anhui/01/2005(H5N1)的HA、NA基因片段,构建质粒pHW2000-HA、pHW2000-NA;以PR8的其它5个内部基因作为骨架,按照1+2+5模式8质粒共转染Vero细胞拯救流感病毒,并对拯救病毒的生物学特性进行检测。结果由Vero拯救出具有高适应生长特性的H5N1亚型人禽流感病毒疫苗株,并检测了重配疫苗株的生物学特性。结论成功从Vero细胞拯救了具有Vero细胞高适应性生长特性的H5N1亚型人禽流感病毒疫苗株,为应用Vero细胞大规模生产流感疫苗奠定了基础。     

重组H9N2禽流感病毒的构建及其在不同细胞中的复制特性

目的利用反向遗传学技术构建具有感染性的H9N2亚型禽流感病毒,并揭示其在不同细胞中的复制特性。方法以A/Quail/Hong Kong/G1/97(H9N2)禽流感病毒基因组RNA为模板,用RT-PCR技术获得该病毒的8条完整基因片段,并分别将它们插入到p HW2000载体上,最终在293T细胞中包装产生重组H9N2病毒。并将重组的H9N2病毒感染不同细胞,观察病毒在不同细胞上的增殖状况来揭示该病毒在不同细胞上的复制特性。结果应用流感病毒8质粒反向遗传学技术,成功获得重组病毒,并揭示了该毒株在不同细胞(A549,MDCK,Vero)中的复制特性,发现A549人肺腺癌细胞更适宜H9N2病毒的正常复制。此外还分析总结了该毒株的与病毒毒力相关的重要位点特征。结论成功建立了H9N2禽流感病毒的反向遗传系统,该系统的建立为在分子水平研究H9N2病毒以及制备H9N2禽流感疫苗提供了依据。     

用8质粒病毒拯救系统产生冷适应减毒的重组A型人流感病毒的研究

目的 利用流感病毒8质粒病毒拯救系统,产生冷适应减毒的重组A型人流感病毒,建立以冷适应流感病毒株为拯救骨架的反向遗传学技术平台.方法 以冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为拯救病毒的骨架,人工合成了该病毒株的6个内部基因片段,即PB2、PB1、PA、NP、M和NS,同时引入5个氨基酸突变作为标签.6个基因片段通过与改造后的转录载体pAD3000连接,构建6个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS.结果 6质粒与PR8的表面基因HA和NA进行"6+2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝活性的冷适应减毒的重组A型人流感病毒,命名为rMDV-A.鸡胚尿囊液中重组病毒的血凝(HA)效价为1∶2 9～1∶2 10.结论 构建的A/Ann Arbor/6/60的6个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发提供了试验材料.     

武装GV1001的重组溶瘤流感病毒构建及抗肿瘤作用研究北大核心CSCD

目的拯救嵌合GV1001的重组溶瘤流感病毒株,并通过体内外实验全面评价其选择性杀伤肝癌的效果。方法将编码GV1001肽的碱基序列串联重复3次序列优化设计后插入到A/PuertoRico/8/34(PR8)病毒的NA片段。采用反向遗传学技术,重组质粒pOV-GV1001-NA与pHW191-PB2、pHW192-PB1、pHW193-PA、pHW194-HA、pHW195-NP、pHW197-M、pHW198-NS PR87个质粒共同转染COS 1/MDCK细胞,拯救得到重组溶瘤病毒rOV-GV1001-NA。经过血凝实验、TCID_(50)、PCR、透射电镜、细胞免疫荧光等鉴定,流式细胞术检测rOV-GV1001-NA诱导肝癌细胞死亡的方式,进一步通过肝癌H22细胞小鼠皮下荷瘤模型评价rOV-GV1001-NA体内溶瘤效果。结果重组溶瘤病毒rOV-GV1001-NA血凝效价为2^(9),病毒滴度达5 LogTCID_(50)/ml,并可在SPF鸡胚中稳定传代;电镜观察形态大小符合流感病毒特点;细胞免疫荧光证明GV1001成功插入并表达;细胞凋亡实验表明rOV-GV1001-NA可选择性诱导肝癌细胞凋亡;肝癌小鼠皮下荷瘤模型证实rOV-GV1001-NA可显著抑制肿瘤生长,延长小鼠生存期,且具有剂量依赖性。结论利用反向遗传学技术成功构建了武装GV1001的重组溶瘤流感病毒,体内外实验表明rOV-GV1001-NA能够靶向杀伤肝癌细胞而对正常细胞无明显影响,该结果将为肿瘤疫苗研究提供新思路,有望为肝癌临床治疗开辟新途径。     

表达PD1抗体的重组溶瘤流感病毒株的构建及效果评价

目的拯救表达免疫检查点PD1抗体的重组流感病毒株,并评价其体内外靶向杀伤肝癌的效果。方法利用反向遗传学技术,选择流感病毒株A/PuertoRico/8/34(PR8)为载体,将免疫检查点抑制剂PD1抗体的重链和轻链分别插入到PR8病毒株PB1、PA基因片段构建重组质粒p Flu-PD1-PB1、p Flu-PD1-PA。构建的质粒与PR8的其余6个内部基因骨架pHW191-PB2、pHW194-HA、pHW195-NP、pHW196-NA、pHW197-M、pHW198-NS基因,转染COS-I和MDCK共培养细胞,通过八质粒病毒拯救系统成功筛选获得重组靶向溶瘤病毒,命名为rFlu-aPD1。通过血凝试验及TCID50测定重组病毒滴度;电镜观察病毒形态;ELISA方法测定PD1抗体含量;MTS方法测定重组病毒对肝癌细胞杀伤效果;流式细胞术检测重组病毒诱导肝癌细胞凋亡效果。利用PDX肝癌小鼠模型,评价重组病毒在体内对肿瘤的抑制效果。结果重组病毒rFlu-aPD1血凝HA效价为2~8～2~9,可在鸡胚中稳定传代;MTS结果显示rFlu-aPD1可选择性杀伤肝癌细胞,而对正常肝细胞无明显影响;重组病毒感染HepG2细胞可检测到较高水平的PD1抗体水平;凋亡实验表明rFlu-aPD1主要通过激活细胞凋亡途径诱导肿瘤细胞死亡。体内实验表明重组病毒可降低PDX肝癌小鼠的肿瘤体积,且动物肿瘤部位的病毒载量显著高于其他组织。结论拯救获得重组溶瘤流感病毒rFlu-aPD1,在体内外均可靶向杀伤肝癌细胞,有望为溶瘤病毒靶向治疗肝癌和肿瘤免疫治疗提供新策略。     

H9N2禽流感病毒反向遗传系统的构建和鉴定

目的 建立H9N2亚型禽流感病毒反向遗传系统,为人禽流感疫苗研制以及传播和致病机制等方面的研究提供技术平台.方法 使用RT-PCR方法获得禽流感H9N2亚型病毒A/Guangzhou/333/99(H9N2)的8条全长基因节段,然后克隆到双表达载体pCI-pol Ⅰ中,获得H9N2禽流感病毒的8个基因节段的8质粒系统.将构建好的8质粒共转染293T细胞后,收获上清接种鸡胚,然后对鸡胚尿囊液进行鉴定;对拯救的病毒进行鉴定.结果 8质粒系统转染293T细胞后可以成功拯救出H9N2禽流感病毒,血凝效价可达到29/50μl,生长特性与野生型病毒类似.结论 成功建立了H9N2禽流感病毒反向遗传系统.     

嵌合人源CTLA4抗体的重组流感病毒选择性杀伤肝癌细胞的研究

目的 拯救嵌合人源免疫检查点细胞毒性T淋巴细胞（huCTLA4）抗体的重组流感病毒株,对其选择性杀伤肝癌细胞的效果进行全面鉴定并评价。方法 通过反向遗传学（reverse genetics,RG）技术,以A/PuertoRico/8/34(PR8)为载体,将编码人源CTLA4(huCTLA4)抗体重链和轻链的基因分别插入到PR8病毒的PB1和PA基因片段,构建重组质粒pFlu-huCTLA4-PB1、p Flu-huCTLA4-PA,与PR8病毒的剩余6个骨架质粒p HW191-PB2、pHW194-HA、pHW195-NP、pHW196-NA、pHW197-M、pHW198-NS共转染COSⅠ和MDCK细胞,经病毒拯救、筛选、鉴定获得重组溶瘤病毒,命名为r Flu-huCTLA4。进一步通过血凝、TCID50测定重组流感病毒滴度;电镜观察病毒形态及大小分布;ELISA法测定huCTLA4抗体含量;结晶紫、MTS方法测定重组病毒对肝癌细胞的选择性杀伤效果;流式细胞术检测重组病毒诱导肝癌细胞死亡方式。利用人源肿瘤异种移植模型肝癌小鼠模型,评价rFlu-huCTLA4在动物体内抗肿瘤作用。...     

H3N2流感病毒体外细胞传代引起的变异

目的 了解用不同来源MDCK细胞分离H3N2流感病毒时,HA和NA片段氨基酸变异情况.方法 选取H3N2流感病毒核酸阳性的咽拭子标本13份,分别在三种不同来源的MDCK细胞中传5代.对标本中病毒及细胞传代的病毒进行HA和NA基因序列测定,分析病毒传代后HA和NA片段变异情况.结果 与咽拭子中的病毒序列相比,MDCK 5代病毒和MDCK-parent 5代病毒均发生了HA和/或NA的突变.9株MDCK 5代病毒HA发生了P221L/P/H突变;8株病毒发生了NA片段的148位或151位的突变.2株MDCK-parent 5代病毒HA221位为多态性.13株病毒MDCK-parent传代后均发生了NA多态性,大多发生在151位.在MDCK-SIAT传5代后的病毒HA均无突变,3株有NA突变.结论 在对流感病毒进行抗原性分析前对病毒进行细胞传代会发生病毒的适应性突变.H3N2流感病毒在MDCK、MDCK-parent中传代后,会引起HA和/或NA突变;MDCK-SIAT传代,病毒变异明显减少.     

用反向遗传技术产生冷适应致弱的重组A型人流感病毒的研究

目的 以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒.方法 对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变.PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体.该重组质粒与PR8进行"7 1"组合的病毒拯救,共转染COS-1细胞.结果 经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒.结论 利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础.     

2009年泉州市H1N1流感监测及基因特性分析

目的 了解2009年泉州地区H1N1流感监测情况,分析泉州市H1N1流感病毒的HA和NA基因特征,探讨该病毒的遗传变异及分子特性.方法 对泉州市H1N1流感监测期间的病人咽拭子采用real-time RT-PCR方法检测病毒核酸,MDCK细胞培养进行病毒分离、鉴定,并提取其中2株代表性毒株病毒RNA;采用RT-PCR扩增病毒HA和NA基因,纯化产物进行核苷酸序列测定;用DNAStar Megalign软件进行序列分析.结果 1020份咽拭子中有200份为H1N1流感病毒核酸阳性,70份季节性流感病毒核酸阳性,其中53份为H3N2亚型,14份为H1N1亚型,3份为B型,并分离到29株甲型H1N1流感病毒株.HA基因经核苷酸序列测定显示,该毒株与北美流行株高度同源,由HA基因核苷酸序列推导的氨基酸系列与疫苗株A/Brisbane/59/2007相比,有22个位于抗原决定簇的氨基酸位点发生变异,但受体结合特异性仍为人样受体.NA基因耐药性位点分析,显示对达菲药物依然敏感.结论 2009年泉州市H1N1流感流行毒株与北美流行株高度同源,相对于疫苗代表株出现了HA蛋白抗原性的改变.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

Reassortment and modification of hemagglutinin cleavage motif of avian/WSN influenza viruses generated by reverse genetics that correlate with attenuation

Avian influenza associated with H9N2 and H5N1 subtypes of avian influenza viruses (AIVs) has raised great concerns in China. To study this problem, reverse genetics has been employed. Three reassortants, rgH9N2, rgH5N1 and rgH5N2, were prepared and compared. Their hemagglutinin (HA) and neuraminidase (NA) genes originated from Chinese AIV isolates of H9N2 or H5N1 subtype, while the rest of their genes were derived from A/WSN/33(H1N1) virus (WSN). In the H5 HA reassortants, the multibasic cleavage site was converted to a monobasic one. The results demonstrated that the reassortants did not produce CPE on MDCK cells in the absence of trypsin, showed egg-adaptation phenotype and stability of HA and NA during consecutive egg passages, and were not lethal to chickens and mice. However, the rgH5N1 reassortant exhibited a residual virulence in terms of lethality to chick embryos and pathogenesis in chickens. It can be concluded that (i) the genetic modification of H5 HA attenuated the H5 reassortants, (ii) the presence of internal WSN proteins contributed to the attenuated properties of the reassortants independently on H5 HA, and (iii) also the overall genome composition contributed to virulence differences. This report provides further contribution of reverse genetics to the knowledge of virulence of influenza viruses.     

Substrate specificity of avian influenza H5N1 neuraminidase

AIM: To characterise neuraminidase(NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography(HPLC) analysis of the enzymatic product. RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked(P 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin(HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid.CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.     

遗传重配获得H5N1流感病毒Vero细胞适应株

目的以传统遗传重配技术选育HSN1流感病毒Veto细胞适应株,制备Vero细胞H5N1流感疫苗。方法以流感病毒Vero细胞适应株A／Yunnan／1／2005Va（H3N2）为母株与反向遗传学技术改造的禽流感病毒疫苗株A／Anhui／1／2005（H5N1）共同感染SPF鸡胚和Vero细胞,用羊抗A／Yunnan／1／2005Va（H3N2）抗体筛选,血抑试验和基因测序鉴定病毒型别,并进行重配株的其他相关生物学试验。结果获得了1株在Vero细胞高产的H5N1流感病毒,重配前后的单价灭活疫苗免疫小鼠抗体血清效价差异无统计学意义（F=0．857,P〉0．05）。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H5N1流感病毒Vero细胞适应株。     

Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece

Summary.  Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996–1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen’s eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996–1997 and 1996–1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996–1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997–1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys. Accepted April 21, 2001 Received     

Human-avian influenza virus reassortants: effect of reassortment pattern on multi-cycle reproduction in MDCK cells

Summary Human-avian influenza reassortants possessing the HA gene of the avian parent virus were tested for their ability to replicate in MDCK cells at 37°C and 31°C. Both avian parent viruses, A/Duck/Ukraine/1/63 (H 3 N 8) and A/Duck/Hoshimin/014/78 (H 5 N 3) induced an efficient multi-cycle infection at 37°C, but replicated poorly at 31°C, whereas the human parent virus, MDCK-adapted variant of A/USSR/90/77 (H 1 N 1) strain, replicated efficiently at both temperatures. The reassortant clone possessing the HA gene of A/Duck/Ukraine/1/63 virus and the other 7 genes of A/USSR/90/77 virus replicated at both temperatures almost as efficiently as the human parent virus. Among the reassortants between A/Duck/Hoshimin/014/78 and A/USSR/90/77, the clones possessing the HA and NA genes of the avian strain, or the HA, NA, NP, and NS genes of the avian strain, and the other genes of the human parent virus, replicated poorly at both temperatures, especially at 31°C, whereas the reassortant possessing the HA, NA, and M genes of the avian virus replicated at both temperatures fairly efficiently. The results are discussed in connection with the limitations imposed by different genes upon avian influenza viruses' ability to replicate in mammalian cells.