Moncayo A C, Medina G M, Kalvatchev Z, Brault A C, Barrera R, Boshell J, Ferro C, Freier J E, Navarro J C, Salas R, De Siger J, Vasquez C, Walder R, Weaver S C
Center for Tropical Disease and Department of Pathology, University of Texas Medical Branch, Galveston 77555-0609, USA.
Am J Trop Med Hyg. 2001 Dec;65(6):738-46. doi: 10.4269/ajtmh.2001.65.738.
During field studies of enzootic Venezuelan equine encephalitis (VEE) viruses associated with epizootic emergence, a large number of virus isolates were made in sylvatic foci of Venezuela and Colombia. To rapidly characterize these isolates, antigenic subtypes were determined by means of immunofluorescence and by single-strand conformational polymorphism (SSCP) analysis by use of an 856-bp fragment from the P62 gene, which we used to distinguish genetic variants. Representative isolates were sequenced to assess the sensitivity of SSCP to detect genetic differences. The SSCP analysis distinguished isolates differing by as little as 1 nucleotide; overall, differences of > or = 1 nucleotide were recognized 89% of the time, and the sensitivity to distinguish strains that differed by only 1 or 4 nucleotides was 17 and 57%, respectively. Phylogenetic analyses of representative sequences showed that all recent isolates from the Catatumbo region of western Venezuela and the middle Magdalena Valley of Colombia were closely related to epizootic subtype IAB and IC strains; strains from Yaracuy and Miranda States were more distantly related. Cocirculation of the same virus genotype in both Colombian and Venezuelan foci indicated that these viruses are readily transported between enzootic regions separated by > 300 km. The SSCP analysis appears to be a simple, fast, and relatively efficient method of screening VEE virus isolates to identify meaningful genetic variants.
在对与动物流行病爆发相关的地方性委内瑞拉马脑炎（VEE）病毒进行野外研究期间，在委内瑞拉和哥伦比亚的野生疫源地分离出了大量病毒。为了快速鉴定这些分离株，通过免疫荧光以及使用来自P62基因的856个碱基对片段进行单链构象多态性（SSCP）分析来确定抗原亚型，我们用该片段来区分基因变异体。对代表性分离株进行测序，以评估SSCP检测基因差异的敏感性。SSCP分析能够区分相差仅1个核苷酸的分离株；总体而言，能在89%的情况下识别出相差≥1个核苷酸的差异，区分仅相差1个或4个核苷酸的毒株的敏感性分别为17%和57%。对代表性序列的系统发育分析表明，来自委内瑞拉西部卡塔通博地区和哥伦比亚马格达莱纳河谷中部的所有近期分离株都与动物流行病亚型IAB和IC毒株密切相关；来自亚拉奎州和米兰达州的毒株关系较远。同一病毒基因型在哥伦比亚和委内瑞拉疫源地的共同传播表明，这些病毒能够在相距300多公里的地方性区域之间轻易传播。SSCP分析似乎是一种简单、快速且相对有效的筛选VEE病毒分离株以鉴定有意义的基因变异体的方法。
