Unidade de Microbiologia Médica/Global Health and Tropical Medicine (GHTM) Research Centre, Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), 1349-008 Lisboa, Portugal.
Microbiol Spectr. 2018 Nov;6(6). doi: 10.1128/microbiolspec.AME-0003-2018.
Viruses, which are the most abundant biological entities on the planet, have been regarded as the "dark matter" of biology in the sense that despite their ubiquity and frequent presence in large numbers, their detection and analysis are not always straightforward. The majority of them are very small (falling under the limit of 0.5 μm), and collectively, they are extraordinarily diverse. In fact, the majority of the genetic diversity on the planet is found in the so-called virosphere, or the world of viruses. Furthermore, the most frequent viral agents of disease in humans display an RNA genome, and frequently evolve very fast, due to the fact that most of their polymerases are devoid of proofreading activity. Therefore, their detection, genetic characterization, and epidemiological surveillance are rather challenging. This review (part of the Curated Collection on Advances in Molecular Epidemiology of Infectious Diseases) describes many of the methods that, throughout the last few decades, have been used for viral detection and analysis. Despite the challenge of having to deal with high genetic diversity, the majority of these methods still depend on the amplification of viral genomic sequences, using sequence-specific or sequence-independent approaches, exploring thermal profiles or a single nucleic acid amplification temperature. Furthermore, viral populations, and especially those with RNA genomes, are not usually genetically uniform but encompass swarms of genetically related, though distinct, viral genomes known as viral quasispecies. Therefore, sequence analysis of viral amplicons needs to take this fact into consideration, as it constitutes a potential analytic problem. Possible technical approaches to deal with it are also described here. *This article is part of a curated collection.
病毒是地球上数量最多的生物实体,它们被认为是生物学中的“暗物质”,因为尽管它们无处不在且大量存在,但它们的检测和分析并不总是那么直接。它们中的大多数都非常小(小于 0.5μm),而且它们的种类极其多样。事实上,地球上大多数的遗传多样性都存在于所谓的病毒圈或病毒世界中。此外,人类中大多数常见的病毒性疾病病原体都具有 RNA 基因组,并且由于它们的大多数聚合酶缺乏校对活性,因此它们经常快速进化。因此,它们的检测、遗传特征分析和流行病学监测具有一定的挑战性。本综述(传染病分子流行病学进展精选集的一部分)描述了过去几十年中用于病毒检测和分析的许多方法。尽管存在应对高度遗传多样性的挑战,但这些方法中的大多数仍然依赖于病毒基因组序列的扩增,使用序列特异性或非序列特异性方法,探索热谱或单一核酸扩增温度。此外,病毒群体,特别是那些具有 RNA 基因组的病毒群体,通常不是遗传上均匀的,而是包含一群遗传上相关但又不同的病毒基因组,这些病毒基因组被称为病毒准种。因此,病毒扩增子的序列分析需要考虑到这一事实,因为这可能构成一个潜在的分析问题。本文还描述了处理这种情况的可能的技术方法。*本文是精选集的一部分。
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