Helton Rebekah R, Wommack K Eric
Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA.
Appl Environ Microbiol. 2009 Apr;75(8):2259-65. doi: 10.1128/AEM.02551-08. Epub 2009 Feb 13.
Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.
直接计数和基因分析表明，水生沉积物中存在丰富多样的病毒群落。到目前为止，尚未有关于河口沉积物病毒多样性在年度周期内的群落生态学分析报告。这种疏忽在很大程度上是由于缺乏用于评估大量环境样本中病毒多样性的分子遗传学方法。在此，随机扩增多态性DNA聚合酶链反应（RAPD-PCR）被用于检测切萨皮克湾沉积物中的病毒基因型多样性。对所有样本使用单一的10聚体寡核苷酸引物，对沉积物病毒群落进行的RAPD-PCR分析在空间和时间尺度上产生了独特的条带模式，特定条带的出现情况在样本集中各不相同。对RAPD-PCR扩增子条带模式的聚类分析表明，沉积物病毒群落随季节变化，且在较小程度上随地理位置变化。对RAPD-PCR扩增子的序列分析显示，76%的沉积物病毒序列与GenBank非冗余蛋白质数据库中的任何序列均无同源性。在与GenBank序列同源的序列中，大多数属于短尾病毒科（24%）和肌尾病毒科（22%）的病毒，这与之前在切萨皮克湾沉积物中观察到的这些形态学科的频率一致。此外，与GenBank非冗余蛋白质序列同源的大多数沉积物病毒序列是噬菌体或原噬菌体（57%）。因此，RAPD-PCR被证明是一种可靠且有用的方法，可用于表征水生沉积物中的病毒群落以及病毒的遗传多样性。
