肠道病毒71型（EV71）AH3株感染性克隆的构建与鉴定

目的 构建肠道病毒71型（Enterovirus71,EV71） AH3株的全基因序列感染性克隆.方法 通过分段扩增和酶切连接将EV71全基因组cDNA片段逐步克隆入PWSK29载体,构建含全病毒基因组序列的重组质粒.转染Vero细胞后,获得拯救病毒.经RT-PCR、酶切、测序、间接免疫荧光（IFA）等方法进行鉴定.结果 酶切鉴定结果与预期一致,序列分析显示为EV71基因;其转染Vero细胞后,可观察到典型的细胞病变;所获得的拯救子代病毒滴度（TCID50）为107.5;IFA检测可见感染细胞出现绿色荧光.结论 成功构建EV71全基因序列感染性克隆,为病毒致病机理及减毒疫苗的研究奠定基础.     

肠道病毒71型的全长cDNA感染性克隆的构建与鉴定

目的构建一株肠道病毒71型的全长cDNA克隆并验证感染性。方法用长片段高保真RT-PCR方法扩增肠道病毒71型87-2008 Xi’an株的cDNA,装入pBR322载体。将该克隆线性化,体外转录后转染RD细胞后,通过观察CPE及RT-PCR验证其感染性。随后利用空斑法测定拯救病毒的一步生长曲线。结果成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆;体外转录及转染RD细胞60 h后可观察到明显的CPE现象;RT-PCR在转染RD细胞中检测到病毒的存在;成功利用空斑法测定了拯救病毒的一步生长曲线,最大滴度达到2×107pfu/mL。结论成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆并验证了其感染性,同时测定了拯救病毒的一步生长曲线,与野生型病毒相近。     

深圳市手足口病重症感染性克隆的构建和分析

目的构建肠道病毒71型（EV71）的感染性克隆,为EV71致病机理的研究和药物的开发建立技术平台。方法选取临床手足口重症儿童粪便样品,经荧光定量PCR检测为阳性,RT-PCR方法扩增出EV71全基因组,通过TA克隆的方法连接到TOPO-XL-PCR载体中,应用T7聚合酶系统将线性化的EV71DNA序列体外转录成RNA,转染人横纹肌肉瘤细胞系RD-A细胞,病毒传代并观察病变,通过间接免疫荧光实验进一步鉴定,获得的感染性克隆进行全基因组测序和序列比对分析。结果 RT-PCR可以获得EV71全长约7.5kb的DNA片段,体外转录并转染后3～5d可观察到典型的肠道病毒致细胞病变,免疫荧光可看到特异标记,序列比对为C4a亚型的EV71病毒。结论构建出具有感染性的EV71全长cDNA克隆,在分子生物学水平上有利于深入研究EV71的致病机制和毒力基因、患者抗体的中和特性、疫苗和药物的效力评估与开发等。     

三种不同转染方法拯救病毒的比较研究

目的比较3种不同转染方法,探索最佳的拯救病毒策略。方法3种转染方法包括：①含T7RNA聚合酶启动子的全长肠道病毒71（EV71）感染性质粒（P—EV71）和T7RNA聚合酶真核表达质粒（VR-1a）共转染Vero细胞;②含T7RNA聚合酶启动子的EV71全长基因片段（PCR产物）与VR-1a共转染Vero细胞;③利用体外转录技术得到EV71病毒全长RNA,直接转染Vero细胞。分别观察3种方法转染后产生细胞病变效应（CPE）时间及病变情况,用RT—PCR扩增拯救病毒核酸,测序验证其正确性,用蛋白印迹方法检测拯救病毒抗原。结果3种方法均可成功转染Vero细胞得到拯救病毒,三种转染方法转染效果比较：方法③优于②、②优于①。结论三种转染方法均可成功拯救EV71病毒,以病毒RNA直接转染效果最好。     

T7RNA聚合酶真核表达质粒的构建及其病毒拯救功能的鉴定

目的 建立T7RNA聚合酶真核表达质粒细胞内病毒拯救系统.方法 利用分子生物学技术,以大肠埃希菌BL21(DE3)的T7RNA聚合酶基因为目的 基因,构建T7RNA聚合酶真核表达质粒VR-1a并鉴定,然后将VR-1a和EV71病毒感染性克隆质粒共转染Vero细胞并传代,观察其细胞病变,同时用RT-PCR检测EV71病毒核酸和用ELISA检测EV71病毒抗原.结果 酶切和测序显示成功构建T7RNA聚合酶真核表达质粒VR-1a,和EV71病毒感染性克隆共转染Vero细胞后出现明显的病变,RT-PCR检测EV71病毒核酸阳性并测序证实,ELISA检测显示有EV71病毒抗原.结论 此方法可以用于细胞内拯救EV71病毒,有望应用于EV71病毒的核酸疫苗免疫研究.     

T7RNA聚合酶真核表达质粒的构建及其病毒拯救功能的鉴定

目的 建立T7RNA聚合酶真核表达质粒细胞内病毒拯救系统.方法 利用分子生物学技术,以大肠埃希菌BL21(DE3)的T7RNA聚合酶基因为目的 基因,构建T7RNA聚合酶真核表达质粒VR-1a并鉴定,然后将VR-1a和EV71病毒感染性克隆质粒共转染Vero细胞并传代,观察其细胞病变,同时用RT-PCR检测EV71病毒核酸和用ELISA检测EV71病毒抗原.结果 酶切和测序显示成功构建T7RNA聚合酶真核表达质粒VR-1a,和EV71病毒感染性克隆共转染Vero细胞后出现明显的病变,RT-PCR检测EV71病毒核酸阳性并测序证实,ELISA检测显示有EV71病毒抗原.结论 此方法可以用于细胞内拯救EV71病毒,有望应用于EV71病毒的核酸疫苗免疫研究.     

T7RNA聚合酶真核表达质粒的构建及其病毒拯救功能的鉴定

目的建立TTRNA聚合酶真核表达质粒细胞内病毒拯救系统。方法利用分子生物学技术,以大肠埃希菌BL21（DE3）的T7RNA聚合酶基因为目的基因,构建TTRNA聚合酶真核表达质粒VR-1a并鉴定,然后将VR-1a和EV71病毒感染性克隆质粒共转染Vero细胞并传代,观察其细胞病变,同时用RT-PCR检测EV71病毒核酸和用ELISA检测EV71病毒抗原。结果酶切和测序显示成功构建T7RNA聚合酶真核表达质粒VR-1a,和EV71病毒感染性克隆共转染Vero细胞后出现明显的病变,RT．PCR检测EV71病毒核酸阳性并测序证实,ELISA检测显示有EV71病毒抗原。结论此方法可以用于细胞内拯救EV71病毒,有望应用于EV71病毒的核酸疫苗免疫研究。     

乙型脑炎病毒活疫苗生产株SA14-14-2的感染性克隆构建

目的　构建乙型脑炎病毒 (乙脑病毒 )活疫苗生产株SA1 4 14 2的感染性克隆。方法　在对SA1 4 14 2株全长基因组测序基础上 ,引入适当酶切位点和T7RNA聚合酶启动子 ,采用分部连接策略 ,获得全长基因组cDNA ,体外转录RNA后 ,转染细胞获得感染性克隆 ,病毒传代培养、抗体中和试验和乳鼠脑腔攻击试验鉴定病毒。结果　由疫苗株cDNA构建了病毒感染性克隆 ,经鉴定为乙脑病毒 ,且病毒毒力比野毒株弱。结论　获得了乙脑病毒活疫苗生产株SA1 4 14 2的感染性克隆 ,为进一步研究疫苗的减毒机理及开发新一代疫苗奠定了基础     

由构建的基因组全长cDNA所回收的我国登革2型病毒株的鉴定

目的 由构建的病毒基因组全长cDNA回收具有感染性的我国登革2型病毒株，为进一步阐明登革2型病毒的致病机制及其新型疫苗的研制奠定基础。方法 用SP6 RNA聚合酶系统制备我国登革2型病毒（D2－43）株基因组全长cDNA的体外RNA转录物，纯化后经电穿孔法转染C6／36细胞，观察致细胞病变作用以判断其感染性。从病变的细胞和培养上清中提取总RNA，通过RT－PCR扩增及克隆测序的方法证实细胞病变确为RNA转录物感染所致。同时收集可产生细胞病变的培养上清，再感染C6／36细胞以进一步证实所回收的登革2型病毒感染的稳定性。结果 以我国D2－43病毒株基因组全长cDNA为模板制备的体外RNA转录物可使C6／36细胞产生病变，从病变细胞和培养上清中可扩增获得病毒特异的基因片段。在培养细胞中进行连续传代仍可产生细胞病变作用。结论 构建的我国D2－43株基因全长cDNA的体外RNA转录物对传代蚊细胞具有感染性，表明可产生完整的病毒颗粒。本研究可为阐明登革2型病毒的致病机理及探索新的预防和治疗措施奠定基础。     

我国登革2、3、4型病毒全长cDNA体外RNA转录体的感染性研究

目的 研究我国登革2、3、4型病毒全长cDNA体外RNA转录体的感染性，为构建登革病毒的感染性克隆、深入阐明病毒的致病机理奠定基础。方法 以我国登革2、3、4型病毒基因组全长cDNA为模板，用SP6 RNA聚合酶系统制备体外RNA转录体，经电穿孔转染细胞，观察其致细胞病变效应。从病变细胞培养上清中提取总RNA，用病毒特异引物进行RT-PCR扩增，以证实细胞病变为转录体RNA的恢复病毒所致。结果 我国登革2、3、4型病毒全长cDNA的体外RNA转录体均可使细胞产生病变，从细胞培养上清中可扩增出登革病毒特异的片段。结论 构建的我国登革2、3、4型病毒全长cDNA的体外RNA转录体具有感染性．可在蚊传代细胞内恢复为登革病毒颗粒。     

肠病毒71型分离株的鉴定及免疫原性研究

目的 在对肠病毒71型(enterovirus 71,EV71)分离株进行鉴定的基础上,对其诱导小鼠产生的免疫应答水平进行研究,拟为进一步的EV71候选疫苗研究奠定基础.方法 超速离心纯化病毒后,采用磷钨酸负染法通过电镜观察病毒形态、大小;采用特异性EV71单抗通过间接免疫荧光法(IFA)检测病毒的特异性;合成EV71特异性引物,通过RT-PCR对病毒进行分子生物学鉴定;病毒的VP1基因序列测定后,与其他EV71序列进行比较,绘制种系发育树,确定病毒基因型;通过腹腔注射途径免疫小鼠,免疫后分离血清通过微量细胞病变抑制法测定血清中和抗体效价,ELISA法检测血清抗体水平和抗体亚型水平.结果 电镜下可观察到典型的圆形病毒颗粒,直径为20～30 nm,呈典型的肠病毒形态;085和087株病毒感染细胞中均能观察到黄绿色荧光,提示该两株病毒可与EV71单抗特异性结合;RT-PCR可以从病毒感染细胞中扩增出226 bp大小的特异性产物带,而采用CA16特异性引物则不能扩增出相应大小的产物带;基于VP1基因序列的种系发育分析显示,087和085株均为C4基因型;085和087株EV71免疫小鼠后可诱导产生能中和包括其本身在内的多个病毒株的中和抗体,针对同一株中和病毒,实验组间差异无统计学意义;针对不同中和病毒株,各实验组中和本株、523-07T株的能力明显高于FY-02T株(P<0.05);ELISA法检测结果显示,接种灭活前后的EV71病毒085和087株后均能诱导小鼠产生抗EV71特异性IgG;IgG亚型为IgG1/IgG2a混合型,灭活病毒免疫组小鼠血清EV71特异性IgG、IgG1、IgG2a水平明显高于活病毒免疫组(P<0.05).两株病毒之间差异无统计学意义.结论 本研究分离到的085和087株病毒均为EV71 C4基因亚型,并具有一定的免疫原性.     

Recovery of duck hepatitis A virus 3 from a stable full-length infectious cDNA clone

Recently, duck hepatitis A virus 3 (DHAV-3) with genetically distinct characteristics from DHAV-1 and DHAV-2 was recognized in South Korea and China. In this short communication, we successfully constructed a stable full-length infectious cDNA clone derived from DHAV-3 by solving instability of cloned full-length cDNA in Escherichia coli (E. coli). The cDNA fragments amplified from the genome of DHAV-3 were assembled and inserted into a low-copy-number plasmid. Finally, a full-length cDNA clone containing an engineered SacII site that served as a genetic marker was obtained. The cDNA clone showed stable by serial passages in E. coli when propagated at 25°C under low level of antibiotic selection. BHK-21 cells were transfected with transcribed RNA from the full-length cDNA clone; infectious viral particles were rescued, showing its fatality to 10-day-old duck embryos. The results indicated that the constructed full-length cDNA clone of DHAV-3 is infectious. By various virological assays, our results indicated that the rescued virus exhibited similar biological properties with the parental virus. Animal experiments revealed that the rescued virus retained the high pathogenicity to 1-day-old ducklings and could induce a fatal hepatitis indistinguishable from its parental virus. Our present studies provide a useful tool for future research on genomic functions and molecular pathogenesis of DHAV-3.     

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.     

Construction and characterization of an infectious cDNA clone of enterovirus type 71 subgenotype C4

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease and induces fatal neurological complications. In recent years, this virus has become a major threat to public health in the Asia–Pacific region, while no effective antiviral therapies and vaccines are currently available. In this study, we constructed and characterized for the first time an infectious full-length EV71 cDNA clone derived from the SHZH98 strain, which was the first subgenotype C4 strain isolated in China. Our data demonstrate that the rescued EV71 viruses exhibited growth kinetics in vitro and morphologies similar to those of the BrCr-TR strain and reached a maximum titer of 10^7.5 TCID₅₀/ml. Although the rescued viruses were able to infect suckling mice, no typical symptoms of EV71 infection were observed for up to 18 days post-inoculation. Taken together our research provides an important tool to study the epidemic strains of EV71 in the Asia–Pacific region and promote the development of vaccines.     

Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region

Non-structural protein 2 (Nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most variable region and postulated to play an important role in cell and tissue tropism of PRRSV. To investigate the role of Nsp2 in the viability and growth of PRRSV in cells in vitro, two cDNA clones were constructed containing a deletion of 63 consecutive nucleotides (pWSK-DCBAd63) or 117 nucleotides (pWSK-DCBAd117) within the Nsp2-encoding region of PRRSV (BJ-4). The clone pWSK-DCBAd63 was infectious and produced viable recombinant virus, whereas clone pWSK-DCBAd117 could not be rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 cells and was stably propagated during sequential in vitro cell passages, like the virus recovered from the full-length cDNA clone of PRRSV BJ-4. In comparison to the parental virus (BJ-4) and the virus recovered from the full-length cDNA clone of the BJ-4 strain, the rescued virus from pWSK-DCBAd63 exhibited enhanced growth kinetics, reaching the peak progeny virus titer by 48 h postinfection. These observations suggest that the Nsp2-encoding region is necessary for productive virus infection, and partial deletion does not influence the viability and propagation of PRRSV in cell culture, which may provide a way to insert a foreign gene into the viral genome as a marker for differentiation. Hanchun Yang and Kyoung-Jin Yoon are equal contribution corresponding authors.