Fraile A, Alonso-Prados J L, Aranda M A, Bernal J J, Malpica J M, García-Arenal F
Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Madrid, Spain.
J Virol. 1997 Feb;71(2):934-40. doi: 10.1128/JVI.71.2.934-940.1997.
Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.
从11个自然种群中采集了217株黄瓜花叶黄瓜病毒（CMV）田间分离株，使用来自CMV菌株I亚组和II亚组基因组RNA的探针，通过核糖核酸酶保护分析（RPA）进行分型。大多数（85%）分析的分离株属于I亚组。对于这些I亚组分离株，在代表RNA1、RNA2以及RNA3中两个开放阅读框的四个探针中，每个探针仅发现两种明显不同的RPA模式，即A和B。基于每个探针的这些RPA模式，定义了不同的单倍型。这些单倍型的频率组成在不同分析种群中有所不同，与采样地点或年份无关。这种遗传结构对应于一个具有局部灭绝和重新定殖的集合种群。大多数I亚组分离株（73%）在所有四个探针上都属于具有RPA模式A（1型）或B（2型）的单倍型。相当一部分I亚组分离株（16%）显示出与这两种主要类型的混合感染迹象，可能由此发生基因交换。观察到通过片段重配发生了基因交换：重配分离株的比例为4%，重配并非随机发生，且重配体在种群中未固定下来。因此，有证据表明I亚组1型和2型分离株之间的重配受到选择抑制。用同时感染1型和2型分离株的植物进行蚜虫传播实验，进一步证明了CMV中重配受到选择抑制。对于RNA3，使用两个RPA探针检测到通过重组发生的基因交换。重组分离株占7%，同样未在CMV种群中固定下来。RNA1、RNA2和RNA3区域的序列分析表明，维持编码序列存在很强的限制，也有证据表明这些限制在1型和2型分化期间以及之后这两种类型的多样化过程中可能有所不同。对编码蛋白进化的限制可能与对基因交换的选择抑制有关。因此，我们的数据不支持当前解释多分体病毒基因组进化以促进基因交换的假说。
