Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes
Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes
复制标题
DOI：
10.1016/j.virol.2003.12.020
复制
发表时间：
2004-04-10
期刊：
影响因子：
3.7
通讯作者：
Buchholz, UJ
中科院分区：
文献类型：
--
作者：
Biacchesi, S;Skiadopoulos, MH;Buchholz, UJ
人类偏肺病毒(HMPV)是新近发现的一种呼吸道疾病的病原体，可引起所有年龄段的人，尤其是婴幼儿。HMPV的特性仍然很差，而且据报道在体外复制效率低下。最近确定了代表两个建议的HMPV基因亚群的两个分离物的完整一致序列(Biacchesi等人，病毒学315(1)(2003)1)。我们已经开发了一个反向遗传系统来产生这些菌株之一，CAN97-83，完全由cDNA组成。我们还恢复了Aversion，rHMPV-GFP，其中增强型绿色荧光蛋白(GFP)是从作为第一个基因插入的转录盒中表达的，N基因的41个核苷酸前导区和前16个核苷酸不受干扰。监测活细胞中GFP表达的能力极大地促进了这种生长缓慢的病毒的初步恢复。此外，从工程转录盒中表达外源基因的能力证实了HMPV转录信号的鉴定，并确认F基因末端信号对于转录终止是高效的。回收在该位置含有外源插入的病毒的能力表明，病毒启动子包含在基因组的Y-末端57nT内。重组HMPV在体外的复制效率与生物来源的HMPV一样高，而rHMPV-GFP的动力学和最终产量降低了数倍。研究了胰酶处理的条件，为提高病毒产量提供了条件。HMPV的另一个版本rHMPV+G1F23被回收，它包含G基因的第二个拷贝和启动子近端的两个额外拷贝，顺序为G1-F2-F3。因此，这个重组基因组将编码11个mRNA，而不是8个，长度为17.3kb，比天然病毒长30%。尽管如此，rHMPV+G1F23病毒在体外复制的效率与rHMPV相比仅略有降低，与rHMPV-GFP基本相同。Northern印迹分析表明，随着F和G基因拷贝数的增加和启动子邻近位置的增加，F和G基因的表达分别增加了6倍和14倍以上，序列分析证实了所添加基因在回收病毒中的完整性。因此，构建含有额外G和F保护性抗原基因拷贝的HMPV疫苗病毒以增加抗原表达或提供额外的HMPV抗原谱系或亚群的表达是可行的。由爱思唯尔公司出版。
Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups (Biacchesi et al., Virology 315 (1) (2003) 1). We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered aversion, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP)was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the Y-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV. Published by Elsevier Inc.