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Laboratory Methods in Molecular Epidemiology: Viral Infections *

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
  • Author: Ricardo Parreira1
  • Editors: Lee W. Riley2, Ronald E. Blanton3
    Affiliations: 1: 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; 2: Divisions of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA; 3: Center for Global Health & Diseases, Case Western Reserve University, Cleveland, OH
  • Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018
  • Received 16 March 2018 Accepted 30 April 2018 Published 02 November 2018
  • Ricardo Parreira, [email protected]
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  • Abstract:

    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.

  • Citation: Parreira R. 2018. Laboratory Methods in Molecular Epidemiology: Viral Infections * . Microbiol Spectrum 6(6):AME-0003-2018. doi:10.1128/microbiolspec.AME-0003-2018.


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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.

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Overview of the viral particle-associated nucleic acid amplification approach. This strategy combines filtration, nuclease treatment, and ultracentrifugation, takes advantage of the size and density differences observed between most viruses and most eukaryotic/prokaryotic cells, and aims at the enrichment of the viral fraction when dealing with complex samples (characterized by the presence of a large amount of nonviral contaminating nucleic acids). The sample is initially homogenized (in the case of solid material), usually by mechanical disintegration and sometimes combined with the use of proteases, followed by the clarification of the homogenate by low-speed centrifugation, allowing removal of nuclei and cellular debris. The homogenate is subsequently filtrated (0.45 or 0.22 μm) to exclude smaller cellular fragments and subcellular organelles (e.g., mitochondria), followed by nuclease treatment for removal of most (though usually not all) nonencapsidated nuclei acids. The nuclease-treated sample is then subjected to ultracentifugation/precipitation steps that concentrate and purify viruses from other contaminants that may still be present. Following a nucleic acid extraction, and a possible conversion of viral RNA to cDNA (in the case of viruses with RNA genomes) by reverse transcription, viral sequences may be amplified and sequenced.

Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018
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Virus chip (microarray). Alignments of multiple virus genomic sequences from different viral families allow the identification of conserved (C) family-specific (1 and 2) regions (C1, C2, C1, and C2) or genome-specific sequences within each viral group (G1 to -5a, G1 to -5b, G1 to -5a, and G1 to -5b). These sequences are represented in the virus microarray as virus-specific and genotype-specific oligonucleotides and are immobilized on a planar solid surface. The identities of the viruses possibly present in different biological samples (specific viruses, multiple viruses, or even recombinant viruses) may be revealed by the obtained pattern of hybridization signals. Both DNA and RNA viruses may be identified (after reverse transcription of their RNA genomes). Unique viruses that are not represented in the array of immobilized oligonucleotides will not be detected.

Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018
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Outline of the SISPA and VIDISCA methods. Viral genomic RNA is converted to cDNA using either tagged (SISPA) or nontagged (VIDISCA) random (6 to 8 nucleotides), poly(A), or virus-specific primers. Second-strand DNA is then synthesized using an 5′-to-3′ exonuclease-free DNA polymerase (usually the Klenow fragment of DNA polymerase I), in the presence of random tagged (SISPA) or untagged random hexa/octamers. dsDNA is amplified by PCR using a single tag-specific primer (SISPA) or digested with restriction endonucleases, followed by the ligation of anchors to the ends of the cleaved dsDNA (VIDISCA). In SISPA, amplified DNA fragments may be processed for either blunt or TA cloning in a vector (followed by Sanger sequencing) or directly sequenced using one of the possible NGS platforms. In the case of VIDISCA, anchor-ligated DNA follows two steps of amplification before it may be used for cloning/Sanger sequencing or NGS analysis. Viruses with DNA genomes may also be readily analyzed by SISPA or VIDISCA. Adapted from references 93 and 107 .

Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018
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MPRCA (also known as rolling-circle amplification [RCA]) and MDA are isothermal DNA amplification methods that explore the polymerization as well as the displacement activities of bacteriophage phi29 DNA polymerase. When used in combination with randomly hybridized primers, both methods support unbiased whole-genome amplification. Viral genomes may be directly amplified (DNA viruses) or first converted to ssDNA (RNA viruses) using reverse transcriptase. Both linear and circular molecules may be amplified by MDA and MPRCA, respectively. Linear DNA molecules (viral ssDNA genomes or cDNA) may also be converted to circular molecules (substrates for MPRCA) using an ssDNA ligase.

Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018
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Techniques used for virus discovery and genomic characterization, and their potentials/limitations

Source: microbiolspec November 2018 vol. 6 no. 6 doi:10.1128/microbiolspec.AME-0003-2018

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