H9N2禽流感病毒反向遗传系统转录/表达载体的构建和验证

设计带有BsmBI、BsaⅠ或AarⅠ酶切位点的引物,用RT PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签.将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3+5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2 AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础.     

利用反向遗传技术产生8基因全禽源流感病毒疫苗候选株

利用反向遗传技术将含有A/Chicken/Shanghai/F/98(H9N2)株禽流感病毒(avian influenza virus,AIV)的6个内部基因与H5N1亚型AIV的2个表面基因HA和NA共转染COS-1细胞,产生了6 2全禽源的重配AIV。将H5N1亚型AIV的HA基因经基因突变致弱,然后将A/Chicken/Shanghai/F/98(H9N2)AIV的6个内部基因的cD-NA和以上致弱的禽源HA基因及NA基因的cDNA分别克隆到转录/表达载体pHW2000中,构建成8个转录/表达质粒。将8个质粒共转染COS-1细胞,24h后收获细胞及上清接种SPF鸡胚,72~90h后鸡胚死亡,收取鸡胚尿囊液进行血凝、血凝抑制试验、序列分析、病毒致病性试验和动物免疫保护试验,最终证实产生了致弱的全禽源AIV疫苗候选株。     

禽流感病毒NS第263~277位核苷酸缺失降低其抗干扰素能力*

2000年以来,多数H5N1亚型禽流感病毒在NS基因的263~277位发生15个碱基的缺失。为了研究此缺失在流感病毒进化中的生物学意义,构建H5N1亚型流感病毒A/SD/04株的HA、NA、NS的全基因表达载体,以及NS基因263~277位删除的突变载体。通过反向遗传学技术,与编码WSN的其他内部基因(PB2,PB1,PA,NP和M)的表达载体进行组合转染,获得在NS基因的263~277位缺失和不缺失的2个重组H5N1亚型流感病毒(RWSN-m248和RWSN-248)。此两个重组病毒在无干扰素产生的Ve     

利用8质粒系统拯救A/Chicken/Shanghai/F/98（H9N2）株禽流感病毒

应用反向遗传技术将含有1998年中国大陆分离株H9N2亚型禽流感病毒(Avianinfluenzavirus,AIV)的8个基因片段的质粒共转染COS_1细胞,产生了与野生病毒生物学特性相同的H9N2亚型AIV。将A Chicken Shanghai F 98(CK SH F 98)株H9N2亚型AIV的8个基因组cDNA分别克隆到polⅠ_polⅡ转录 表达载体pHW2 0 0 0中,构建成8个转录表达载体重组质粒。将这8个质粒共转染COS_1细胞,2 4h后收获细胞及上清接种SPF鸡胚,4 8h后收取鸡胚尿囊液继续进行鸡胚传代,产生能致死鸡胚的病毒。经血凝、血凝抑制试验、序列分析和电镜观察,证实产生了CK SH F 98(H9N2 )株AIV。     

H3亚型鸭源流感病毒聚合酶PB1基因的序列分析

目的阐明H3亚型鸭流感病毒与其他亚型流感病毒的关系。方法对活禽市场分离的3株H3N8亚型鸭源流感病毒聚合酶PB1基因进行了序列分析。结果3株鸭源H3N8流感病毒聚合酶PB1基因核苷酸同源性为99.9%,与H9N2亚型流感病毒(DK/ST/2143/00)的同源性为96.31%~96.44%,而与H3N8亚型鸭流感病毒(Mal/Alberta/279/98)为88.65%~88.79%。系统进化树分析表明,本实验中的3株病毒属于相同的分支,且与A/duck/Hong Kong/Y439为代表的H9N2亚型禽流感病毒位于一进化分支,说明三株H3N8亚型流感病毒重排了H9N2亚型禽流感病毒的基因片段。结论不同亚型禽流感病毒在贮存宿主体内的重排以及重排病毒的新特点如鸭H3N8亚型流感病毒对禽的致病性,应当引起我们的高度重视。     

2006～2007年流行流感病毒减毒疫苗株的构建

选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为骨架病毒,对其6个内部基因片段进行了全基因合成,同时人工引入5个氨基酸突变(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA来源于2006-2007当年流行株A/New Caledonia/20/99(H1N1).8个基因片段通过与改造后的转录载体pAD3000连接,构建8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、pMDV-A-HA、pMDV-A-NA.6质粒与当年流行株的表面基因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性的冷适应减毒的重组A型人流感病毒.鸡胚尿囊液中重组病毒的血凝效价为l:279-1:210.构建的A/AA/6/60 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奠定了基础.     

用反向遗传操作技术产生致弱的H5亚型重组流感病毒

选择一株鹅源H5N1亚型禽流感病毒 (AIV) ,缺失其HA基因裂解序列的 4个碱性氨基酸、使HA裂解模式由高致病性的PQRERRRKKR↓GL突变为低致病性的PQRESR↓GL ,将修饰的HA基因克隆入转录 表达载体pHW2 0 0 0、构建质粒pHW5 2 4_HA ,将该毒株和H9N2亚型毒株的NA全基因分别克隆入pHW2 0 0 0 ,构建质粒pHW5 0 6_NA和pHW2 0 6_NA。将pHW5 2 4_HA与pHW5 0 6_NA或pHW2 0 6_NA组合、均用A WSN 33(H1N1)提供 6个内部基因 ,两个组合的 8个质粒分别共转染COS_1细胞 ,产生了H5N1和H5N2两个亚型的基因重排病毒。通过在鸡胚中的连续传代和适应 ,2个重组病毒血凝价上升到 1∶2 9、表面基因稳定、对 6周龄SPF鸡不表现致病性 ,H5N2重组病毒对鸡胚的毒力低于H5N1病毒。这种尝试证明反向遗传操作技术是研究AIV致病性和构建疫苗候选株的有用工具     

神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性的影响

近年来H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)茎部15~20个氨基酸的自发缺失时有报道,突变对于AIV生物学特性的影响还没有得到系统研究。应用反向遗传操作技术,拯救获得5株具有不同NA茎部长度的H5N1/PR8重组AIV。重组病毒的内部基因和血凝素(HA)基因来源相同,NA基因来源不同,并在NA茎部进行20个氨基酸的删除或添加突变。通过研究其生物学特性发现,5株重组病毒在SPF鸡胚中繁殖良好,其EID50、MDT和平均病毒滴度相似;NA茎部长短影响病毒的解凝能力,长茎病毒红细胞解脱能力比短茎病毒强;NA茎部15或20个氨基酸删除突变提高了重组病毒在MDCK细胞上的繁殖能力,短茎病毒释放出的病毒粒子数量是长茎病毒的10~100倍,释放时间提前6~10h,短茎病毒在MDCK细胞上形成的空斑也明显比长茎病毒的空斑大。实验结果揭示了AIV NA茎部氨基酸缺失突变的生物学意义,NA茎部15或20个氨基酸删除突变增强了AIV的细胞适应性,可能与现阶段H5N1亚型AIV宿主范围进一步扩大有关。利用反向遗传技术成功拯救了5株H5N1/PR8重组流感病毒,为流感病毒基因功能研究和重组疫苗研究建立了技术平台。通过对AIV NA茎部氨基酸的删除突变提高了病毒在MDCK细胞上的繁殖产量,为流感病毒细胞苗的生产提供了新的思路。     

利用反向遗传技术研究H9N2亚型AIV传播途径的分子机制

利用反向遗传技术,通过基因重排方法,产生两个表面基因来自A/Chicken/Guangdong/SS/94(H9N2)禽流感病毒(avian influenza virus,AIV)株和其余基因来自A/Chicken/Shanghai/F/98(H9N2)AIV株的3株H9N2亚型重排AIV,动物传播性试验发现A/Chicken/Shanghai/F/98(H9N2)株、A/Chicken/Guangdong/SS/94(H9N2)AIV株和3株H9N2亚型重排AIV都可以经直接接触途径传播;在粪便接触途径下,3株重排AIV都不经粪便接触传播;只有A/Chicken/Shanghai/F/98(H9N2)株和重排AIV RF7/SSHA能经过气溶胶途径传播。HI试验结果进一步证明了以上的结果。实验结果表明H9N2亚型AIV的NA基因与H9N2亚型AIV气溶胶传播途径有重要的关系,即1998年中国大陆H9N2亚型AIV大流行可能是因为病毒获得气溶胶传播途径的特性,推测病毒的NA基因发挥了重要作用。     

禽流感病毒NS第263～277位核苷酸缺失降低其抗干扰素能力

2000年以来,多数H5N1亚型禽流感病毒在NS基因的263～277位发牛15个碱基的缺失。为了研究此缺失在流感病毒进化中的生物学意义,构建H5N1亚型流感病毒A／SD／04株的HA、NA、NS的全基因表达载体,以及NS基因263～277位删除的突变载体。通过反向遗传学技术,与编码WSN的其他内部基因（PB2,PB1,PA,NP和M）的表达载体进行组合转染,获得在NS基因的263～277位缺失和不缺失的2个重组H5N1亚型流感病毒（RWSN—m248和RWSN-248）。此两个重组病毒在无干扰素产生的Vero细胞上的繁殖滴度相似,在能产生干扰素的细胞MDCK和COS-1细胞上的繁殖滴度有明显差异。两个重组病毒在鸡胚中的繁殖滴,IVPI,MDT和EID50均无显著差异。说明NS基因的263～277位核苷酸的缺失不影响病毒的整体毒力,但降低了H5N1的抗干扰素能力。     

重组细胞高产型H3N2猪流感病毒株的拯救

为拯救出一株能够在动物传代细胞中高水平复制的H3N2亚型猪流感疫苗株,利用反向遗传操作技术,将A/Goose/Dalian/3/01(H9N2)毒株的PB1、PA、NP、M、NS基因和A/PR/8/34毒株的PB2基因作为内部基因与猪流感病毒A/Swine/Henan/S4/01(H3N2)毒株的HA、NA基因进行重组,成功拯救出了具有高度细胞适应性毒株rH3N2株,该毒株接毒MDCK细胞60h后,血凝价可以达到1∶512,表明该毒株具有高度适应细胞繁殖特性,为H3N2亚型猪流感病毒细胞培养型疫苗的研制奠定了基础。     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

H5N2 avian influenza outbreak in Texas in 2004: the first highly pathogenic strain in the United States in 20 years?

In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.     

A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry.     

Characterization of Influenza A (H7N9) Viruses Isolated from Human Cases Imported into Taiwan

A novel avian influenza A (H7N9) virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave) was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave). The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.     

H5N1亚型禽流感病毒基因重排后毒力的变化

[目的]为了探讨高致病性禽流感病毒对水禽致病性差异的分子致病机理.[方法]我们对从野鸭分离到的H5N1亚型禽流感病毒的生物学特性进行鉴定,其中A/mallard/Huadong/Y/2003(Y)是对麻鸭无致病性病毒,而 A/mallard/Huadong/S/2005(S)是对麻鸭高致病性病毒.利用反向遗传技术构建一系列单个和多个基因组合替换基因重排病毒,并验证重排病毒在麻鸭上的致病力.[结果]研究表明,PB2, PB1, PA(3P), HA单基因以及3P基因组合替换的使S病毒对麻鸭的毒力完全致弱,但相应的基因替换后仅使Y病毒对麻鸭的毒力略有上升.两病毒的其它基因对毒力影响较小.[结论]H5N1亚型禽流感病毒对麻鸭的致病力受多基因调控,且这种调控作用在不同病毒骨架上的影响不一致,强毒受影响程度远比弱毒的大.     

H9N2亚型禽流感病毒基因组全长序列测定和各基因的遗传分析

用RTPCR技术及cDNA末端快速扩增法获得禽流感病毒分离株A/Chicken/Shanghai/F/98（H9N2）代表基因组全长的8个基因片段。基因组序列比较及遗传进化分析结果表明,Chicken/Shanghai/F/98的8个基因均不属于Quail/Hong Kong/G1/97亚系,与香港禽流感事件没有直接关系。它与Chicken/Beijing/1/94的HA、NA、M、NS基因同源率分别为96.7%、96.4%、97.5%和98.0%,这4个基因属于Chicken/Beijing/1/94亚系,其中,NA基因与Duck/Hong Kong/Y280/97的同源率为97.4%,而且它们均在205位后缺失9个核苷酸。而PB2、PB1、PA和NP基因与已知的3个亚系关系较远,分别在相应的进化树上另成分支。因此,Chicken/Shanghai/F/98是两个以上不同基因亚系间发生自然重排的产物。     

Identification of potential virulence determinants associated H9N2 avian influenza virus PB2 E627K mutation by comparative proteomics

Some highly pathogenic H5N1, H7N9, and H10N8 isolated from China carried six internal genes from H9N2 avian influenza viruses (AIV) and the key amino acids at 627 in PB2 of these viruses had mutated to K. To investigate the mechanism of increased pathogenicity for H9N2 AIV PB2 627K, we analyzed the difference in mouse lung proteins expression response to PB2 K627E. By iTRAQ method, we found that the mutated K627E contributed to a set of differentially expressed lung proteins, including five upregulated proteins and nine downregulated proteins at 12 h postinfection; ten upregulated proteins and 25 downregulated proteins at 72 h postinfection. These proteins were chiefly involved within the cytoskeleton and motor proteins, antiviral proteins, regulation of glucocorticoids signal‐associated proteins, pro‐ and anti‐inflammatory proteins. Alteration of moesin, FKBP4, Hsp70, ezrin, and pulmonary surfactant protein A (sp‐A) may play important roles in increasing virulence and decreasing lungs antiviral response. Further, three upregulated proteins (moesin, ezrin, and sp‐A) caused by PB2 K627E were also confirmed in A549 cells. Moreover, overexpression of sp‐A in A549 inhibited virus replication and downregulation promoted virus replication. In this study, sp‐A as a potential virulence determinant associated H9N2 AIV PB2 E627K mutation was identified using comparative proteomics.     

H9N2亚型禽流感病毒基因组的遗传进化分析

2009～2011年从江苏省、湖北省和安徽省等地来源于鸡、鸭、鹌鹑和鸽子的样品中分离鉴定出16株H9N2亚型禽流感病毒。通过反转录聚合酶链式反应(RT-PCR)扩增出分离株的全基因片段,并对其进行测序及遗传进化分析。序列分析显示,16株病毒HA基因裂解位点氨基酸序列为P-S-R/K-S-S-R,符合低致病性禽流感的分子特征;226位均为L,具有与哺乳动物唾液酸α,2-6受体结合的特性。M2基因均出现了对金刚烷胺产生耐药性的N31S突变。不同宿主来源的H9亚型AIV的主要分子特征一致。全基因遗传进化分析表明16株H9N2亚型禽流感病毒全基因发生了3配体重组,即以F98亚系AIV为骨架,HA来源于Y280亚系,PB2和M基因来源于G1亚系,形成了2种新的基因型。因此,要加强对H9N2亚型禽流感病毒的监测,密切关注它的重组趋势。