Biacchesi Stéphane, Skiadopoulos Mario H, Tran Kim C, Murphy Brian R, Collins Peter L, Buchholz Ursula J
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-8007, USA.
Virology. 2004 Apr 10;321(2):247-59. doi: 10.1016/j.virol.2003.12.020.
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. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, 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 3'-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.
人偏肺病毒（HMPV）是一种最近才被认识到的呼吸道疾病病原体，可感染各年龄段人群，尤其是婴幼儿。HMPV的特征仍不太明确，据报道其在体外复制效率较低。最近已确定了代表两个拟议的HMPV遗传亚组的两个分离株的完整共有序列。我们开发了一种反向遗传系统，能够完全从cDNA产生其中一个分离株CAN97-83。我们还获得了一个版本rHMPV-GFP，其中增强型绿色荧光蛋白（GFP）由作为第一个基因插入的转录盒表达，N基因的41个核苷酸的前导区和前16个核苷酸未受干扰。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回收。此外，从工程化转录盒表达外源基因的能力证实了HMPV转录信号的鉴定，并确定F基因末端信号对转录终止非常有效。在该位置回收含有外源插入片段的病毒的能力表明病毒启动子包含在基因组3'末端的57个核苷酸内。重组HMPV在体外的复制效率与生物学来源的HMPV一样高，而rHMPV-GFP的动力学和最终产量降低了几倍。研究了胰蛋白酶处理条件，提高了病毒产量。获得了另一个HMPV版本rHMPV+G1F23，它包含G基因的第二个拷贝和F基因的两个额外拷贝，按G1-F2-F3的顺序位于启动子近端位置。因此，这个重组基因组将编码11个mRNA而不是8个，长度为17.3 kb，比天然病毒长30%。尽管如此，rHMPV+G1F23病毒在体外的复制效率与rHMPV相比仅略有降低，与rHMPV-GFP基本相同。Northern印迹分析表明，添加的F和G基因拷贝数量增加且位于启动子近端，导致F和G mRNA的表达分别增加了6倍和14倍以上，序列分析证实回收病毒中添加基因的完整性。因此，构建一种含有额外拷贝的G和F推定保护性抗原基因的HMPV疫苗病毒以增加抗原表达或提供HMPV其他抗原谱系或亚组的代表性应该是可行的。
