Lemon Ken, Nguyen D Tien, Ludlow Martin, Rennick Linda J, Yüksel Selma, van Amerongen Geert, McQuaid Stephen, Rima Bert K, de Swart Rik L, Duprex W Paul
School of Medicine, Dentistry and Biomedical Sciences, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.
J Virol. 2015 Mar;89(5):2849-56. doi: 10.1128/JVI.03587-14. Epub 2014 Dec 24.
Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.
Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.
人呼吸道合胞病毒（HRSV）是婴儿严重呼吸道疾病最重要的病毒病因。已鉴定出两个亚组（A和B），它们在年度流行期间共同传播或交替出现，引起难以区分的疾病。现有的HRSV体外和体内模型几乎完全集中于A亚组病毒。在此，基于直接从一名住院婴儿的未经传代的临床标本获得的共有基因组序列，产生了一种重组（r）B亚组病毒（rHRSV(B05)）。在基因组的磷蛋白和基质基因之间（位置5）引入了一个包含编码增强型绿色荧光蛋白（EGFP）基因的额外转录单元，以产生rHRSV(B05)EGFP(5)。重组病毒在HEp-2细胞和在气液界面生长的高度分化的正常人支气管细胞中均能高效复制。棉鼠（棉鼠属）经鼻内感染后，在鼻中隔和鼻甲的上皮细胞中产生大量EGFP(+)细胞。当以相对大的接种量给药时，该病毒在细支气管上皮细胞中也能高效复制，并在上呼吸道和下呼吸道广泛传播。在气管的纤毛上皮细胞中未观察到病毒复制。这是首个具有当前流行的野生型病毒基因组成的强毒力rHRSV毒株。在无细胞病变变化的情况下，通过EGFP荧光对感染细胞进行体内追踪，提高了HRSV发病机制研究中病毒检测的灵敏度。
病毒学作为一门学科一直依赖于监测体外病毒培养后的细胞病变效应。然而，从患者分离的野生型病毒通常不会对感染细胞造成显著变化，因此需要盲目传代。这可能导致基因和表型变化，并产生在疾病动物模型中毒力减弱的高滴度、实验室适应病毒。为解决这一问题，我们从直接取自感染婴儿的样本中确定了一株未经传代的人呼吸道合胞病毒的基因组序列，组装了一个分子克隆，并获得了一种野生型重组病毒。添加编码增强型绿色荧光蛋白的基因使得该野生型病毒能够在原代人细胞和活体动物中在无显著细胞病变效应的情况下被追踪。荧光细胞成像被证明是监测病毒传播的一种非常有价值的工具，可能有助于改进评估新型干预策略的检测方法。
