用反向遗传技术致弱基因VIId型鹅源新城疫病毒ZJI株

将新城疫病毒ZJI株基因组cDNA全长分成7个片段,依次连接并克隆至TVT7R转录载体中,构建了含ZJI株全基因组cDNA的转录载体(pNDV/ZJI),pNDV/ZJI与3个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的新城疫病毒粒子。设计两对引物,经overlapPCR方法将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸后,替换pNDV/ZJI上的对应序列,构建了转录载体pNDV/ZJIFM,将pNDV/ZJIFM与3个辅助表达质粒共转染BSR-T7/5细胞,成功拯救出了致弱的基因VIId型鹅源新城疫病毒NDV/ZJIFM,获救病毒的鸡胚最小致死剂量平均死亡时间(MDT)大于120h,同时该病毒的脑内接种致病指数(ICPI)为0.16,上述结果表明,获救病毒的毒力已被致弱,是一个较为理想的疫苗候选株。     

新城疫病毒抗肿瘤效应机制的研究现状

溶瘤病毒疗法是一种重要的抗癌手段。经研究,新城疫病毒(Newcastlediseasevirus,NDV)是一种非常有效的溶瘤病毒(oncolyticvirus,OV),它能选择性杀伤肿瘤细胞,对正常细胞几乎无影响。本文从NDV诱导肿瘤细胞发生凋亡、自噬、抑制细胞代谢、刺激机体免疫反应和诱导肿瘤细胞发生核糖体应激反应等方面综述了新城疫病毒的抗肿瘤效应机制,并着重探讨了NDV通过诱导核糖体压力应激反应调控肿瘤细胞翻译系统并诱导细胞发生凋亡的具体机制,旨在为今后NDV抗肿瘤作用的深入研究及靶向治疗癌症提供更加扎实丰富的理论基础。     

Comparison of RNA polymerase associated with Newcastle disease virus and a temperature-sensitive mutant of Newcastle disease virus isolated from persistently infected L cells.

An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo) and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.     

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

Background

Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.

Methods

An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.

Results

For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 10¹ 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.

Conclusion

The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

     

应用新城疫病毒治疗肿瘤的研究进展

新城疫病毒可以特异地杀伤肿瘤细胞,而对正常细胞没有伤害,目前在临床实验中认为是安全、有效的溶瘤试剂。随着近年来反向遗传操作技术的日趋成熟,该技术开始应用到新城疫病毒溶瘤效果的优化方面,通过改造新城疫病毒的F基因,及表达重组粒细胞巨噬细胞集落刺激因子,干扰素-γ,白细胞介素-2和肿瘤坏死因子-α等肿瘤杀伤因子,使该病毒具备更加优越的肿瘤杀伤能力,成为肿瘤治疗领域一个新兴的亮点,为癌症的临床治疗提供了崭新的前景。以下将简要介绍应用反向遗传操作技术重组新城疫病毒优化肿瘤治疗效果的研究进展,以及本实验室在相关领域的研究情况。     

Expression of a cloned hemagglutinin-neuraminidase gene from Newcastle disease virus in murine myeloma cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin-neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoid fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.     

Characterization of an alternate form of Newcastle disease virus fusion protein

The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes and infected COS-7 cells (J. Virol. 77:1951-1963, 2003). One form is the classical type 1 glycoprotein, while the other is a polytopic form in which approximately 200 amino acids of the amino-terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein-expressing cells, and antibodies specific for these sequences inhibited red blood cell fusion to hemagglutinin-neuraminidase and F protein-expressing cells, suggesting a role for surface-expressed CT sequences in cell-cell fusion. Extending these findings, we have found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of the F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single-cycle infections, as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed.     

Pathotyping of Newcastle disease virus with a filamentous bacteriophage

A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV). In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed. This method can differentiate the velogenic strains from the mesogenic and lentogenic strains. An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel). Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains. These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.     

新城疫病毒FMW株体外溶瘤作用及其机制分析

[目的]筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫(New Castle disease virus,NDV)毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础.[方法]以体外噻唑蓝法测定NDV对A549、SMMC7721等肿瘤细胞及人胚干细胞L-02、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数.利用形态学观察、Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制.[结果]从近50株NDV中筛选出NDV-FMW,以20 MOI病毒作用A549、SMMC7721等肿瘤细胞48 h,细胞生长抑制率达60%,NDV-FMW诱导肿瘤细胞发生凋亡,效应呈时间和剂量的依赖性,凋亡细胞出现核染色质断裂、浓缩及二倍体亚峰,细胞周期阻滞于GO/G1期,此外,病毒感染A549细胞16 h后开始检测到活化的Caspase-3裂解片段及PARP裂解大片段.[结论]NDV-FMW株体外能高效抑制肿瘤细胞的增殖,并经Caspase-3途径诱导肿瘤细胞凋亡.FMW株具有自主知识产权,其良好的体外溶瘤能力为进一步探讨体内抗肿瘤及临床试验的进行奠定了基础,并有可能为恶性肿瘤的治疗提供新的生物制剂.     

新城疫病毒FMW株体外溶瘤作用及其机制

摘要：【目的】筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫（New Castle disease virus, NDV）毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础。【方法】以体外噻唑蓝法测定NDV对A549、 SMMC7721等肿瘤细胞及人胚干细胞L-O2、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数。利用形态学观察、 Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制。【结果】从近50株NDV中筛选出NDV-FMW,以     

Complete genome sequencing and analysis of an anti-tumor Newcastle disease virus strain

HBNU/LSRC/F3, a Newcastle disease virus (NDV) strain stored in our lab, exhibited an anti-tumor ability in our previous studies. Nonetheless, very little is known about its genome sequence, which is vital for further study. Here, the complete HBNU/LSRC/F3 genome was fully sequenced and compared with other NDV sequences. Its genome contained 15,192 nucleotides (nt) consisting of two termini and six genes in the following order: 3′-Le-NP-P-M-F-HN-L-Tr-5′. Phylogenetic analysis indicated that this NDV strain belonged to the Class II genotype IX group. A multibasic amino acid (aa) sequence was found at the cleavage site (¹¹²RRQRR↓F¹¹⁷) within the fusion (F) protein, and a 6 nt insertion was present in the 5′ non-coding region of the NP gene. The whole genome sequence was highly similar to other genotype IX NDV genomes reported in China. Overall, this study provides insight into the sequence characteristics of genotype IX NDVs, which will be useful for subsequent investigations.     

Newcastle disease virus and Chlamydia psittaci in free-living raptors from eastern Germany

Organ samples from free-living raptors from the federal states of Berlin and Brandenburg in eastern Germany were tested for Newcastle disease virus (NDV; n = 331) and Chlamydia psittaci (n = 39) by polymerase chain reaction (PCR). In 18 individuals NDV nucleic acids were detected. These samples originated from barn owls (Tyto alba; n = 15, 28%), tawny owl (Strix aluco; n = 1, 5%), common buzzard (Buteo buteo, n = 1, 1%), and European kestrel (Falco tinnunculus; n = 1, 4%). In 29 (74%) of 39 samples C. psittaci was detected. Chlamydia psittaci is common in free-living birds of prey in the investigated area.     

利用反向遗传操作技术产生ZJI株鹅源新城疫病毒

利用反向遗传操作技术,将ZJI株鹅源新城疫病毒全基因组cNDA克隆(NDV3GM122)和含该毒株NP、P及L基因的3个表达载体(pCI-NP、pCI-P与pCI-L)共转染BSR-T7/5细胞;同时,将NDV3GM122与含新城疫病毒La Sota毒株NP、P及L基因的3个表达载体(pCIneoNP、pCIneoP与pCIneoL)进行共转染。通过间接免疫荧光实验(Indiectimmunofluorescence assay,IFA)以及接种鸡胚后进行血凝(Hemagglutinin,HA)与血凝抑制(Hemagglutinininhibition,HI)试验、RT-PCR扩增和电镜观察,结果均证实全基因组cDNA克隆NDV3GM122与La Sota毒株表达载体共转染组产生了有血凝性的鹅源新城疫病毒,而NDV3GM122与ZJI株表达载体共转染组暂未检测到有血凝性的病毒。ZJI株鹅源新城疫病毒的拯救成功为对该病毒进行功能基因组研究和疫苗的研制等后续工作打下了基础。     

Newcastle disease virus in Taiwan: II, Relationship between polykaryocytosis and virus virulence

Fifteen selcted local isolates and five known Newcastle disease virus strains were examined for their cytopathic effects in chick embryo kidney (CEK) cells, egg-infectious units in chick embryos (CE), virulence by mean death time, intracerebral and intravenous pathogenicity indexes for CE and chicks, and ability to cause polykaryocytosis of fusion from within (FFWI) or fusion from without (FFWO) in CEK and BHK-21 monolayer cells. The capacity of the different virus strains to induce cell FFWI at 15 hr post-infection was related to their virulence for CE and chicks, but cell FFWO did not seem to be any relationship with the virulence of the strains.     

Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.

A cDNA copy of the RNA encoding the fusion (F) protein of Newcastle disease virus (NDV) strain Texas, a velogenic strain of NDV, was obtained and the sequence was determined. The 1,792-base-pair sequence encodes a protein of 553 amino acids which has essential features previously established for the F protein of virulent NDV strains. These include the presence of three strongly hydrophobic regions and pairs of dibasic amino acids in the pentapeptide Arg-Arg-Gln-Arg-Arg preceding the putative cleavage site. When inserted into a fowlpox virus vector, a glycosylated protein was expressed and presented on the surface of infected chicken embryo fibroblast cells. The F protein expressed by the recombinant fowlpox virus was cleaved into two polypeptides. When inoculated into susceptible birds by a variety of routes, an immunological response was induced. Ocular or oral administration of the recombinant fowlpox virus gave partial protection, whereas both intramuscular and wing-web routes of inoculation gave complete protection after a single inoculation.