Firestone C Y, Whitehead S S, Collins P L, Murphy B R, Crowe J E
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0720, USA.
Virology. 1996 Nov 15;225(2):419-22. doi: 10.1006/viro.1996.0618.
The complete nucleotide sequence of the RSV cpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passaged cp-RSV, and cpts-248 virus, which constitute the series of progenitor viruses. RSV cpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36 degrees) than its cpts-248 parent virus (shut-off temperature 38 degrees) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited by cp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for the ts and attenuation phenotypes of its chemically mutagenized derivatives, cpts-248 and cpts-248/404. Compared with its cpts-248 parent, the cpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, the cpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of the cpts-248/404 in tissue culture. Biologically cloned derivatives of RSV cpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of the cpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans.
呼吸道合胞病毒cpts - 248/404减毒活疫苗候选株的完整核苷酸序列由克隆的cDNA测定，并与呼吸道合胞病毒A2/HEK7野生型、冷传代cp - RSV和cpts - 248病毒的序列进行比较，这些构成了该系列的原始病毒。与它的cpts - 248亲本病毒（关闭温度38摄氏度）相比，呼吸道合胞病毒cpts - 248/404的减毒程度更高且温度敏感性更强（ts）（关闭温度36摄氏度），目前正在进行人体I期临床试验评估。我们的最终目标是确定cp - RSV表现出的宿主范围减毒表型（即在组织培养中高效复制但在黑猩猩和人类中复制减少）以及其化学诱变衍生物cpts - 248和cpts - 248/404的ts和减毒表型的遗传基础。与它的cpts - 248亲本相比，cpts - 248/404病毒在聚合酶（L）蛋白中有一个氨基酸变化，在M2基因起始序列中有一个单核苷酸替换。总体而言，cpts - 248/404突变体与其野生型呼吸道合胞病毒A2/HEK7原始株在七个氨基酸上存在差异[聚合酶（L）蛋白中有四个，融合（F）糖蛋白中有两个，核蛋白（N）中有一个]，并且在M2基因起始序列中有一个核苷酸差异。在cpts - 248/404在组织培养传代过程中，vRNA前导区核苷酸位置4（负链，G或C，与呼吸道合胞病毒A2/HEK7原始株中的G相比）出现了异质性。在位置4存在差异的呼吸道合胞病毒cpts - 248/404病毒的生物学克隆衍生物具有相同水平的温度敏感性，并且在小鼠的上呼吸道和下呼吸道中表现出相同水平的复制，这表明该位置的异质性在临床上不相关。确定cpts - 248/404病毒的核苷酸序列将有助于评估在疫苗生产过程中以及在人体中复制后与减毒表型相关的八个突变的稳定性。
