Lu Z, Rezapkin G V, Douthitt M P, Ran Y, Asher D M, Levenbook I S, Chumakov K M
Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852, USA.
J Gen Virol. 1996 Feb;77 ( Pt 2 ):273-80. doi: 10.1099/0022-1317-77-2-273.
Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU-->AU or GU-->GC substitution at a complementary pair formed by nucleotides 480 and 525, U-->C substitution at nucleotide 612, and GU-->AU or GU-->GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral 'passengers', passively co-selected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88-->Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production.
减毒脊髓灰质炎病毒株的复制会导致其部分毒力恢复。最近，我们在细胞培养物中发现了Sabin 1脊髓灰质炎病毒中积累的突变。在此，我们报告该病毒在猴子中枢神经系统（CNS）复制过程中发生的基因变化。对从恒河猴（接种到脊髓）CNS不同部位分离的病毒进行序列异质性筛选，并使用PCR和限制性内切酶切割突变分析（MAPREC）对新发现的突变进行独立确认和定量。本研究中确定的所有持续积累的突变均位于非翻译区：核苷酸480和525形成的互补对处的GU→AU或GU→GC替换、核苷酸612处的U→C替换，以及紧接在poly(A)尾之前的核苷酸7427/7441形成的碱基对的GU→AU或GU→GC替换。除一个突变（7427）外，所有这些突变先前在疫苗接种者的细胞培养传代或粪便分离物中已被鉴定。对11株CNS分离株的测序还鉴定出一些随机沉默突变，这些突变作为中性“乘客”积累，与同一RNA分子上真正可选择的突变被动共选择。一株分离株在核苷酸2741处也含有野生型碱基（VP1中的Ala88→Thr）。我们的结果表明Sabin 1脊髓灰质炎病毒在猴子CNS中具有显著的遗传稳定性，这表明毒力恢复是由非常有限数量的突变决定的。这些突变可以通过MAPREC进行检测，以监测口服脊髓灰质炎疫苗（OPV）生产的一致性。
