Principles and Practice of Clinical Virology is the bible for all working in the field of clinical virology – from the trainee to the expert because. download Textbook of Medical Virology - 1st Edition. Print Book & E-Book. DRM-free (EPub, PDF, Mobi) Some of the topics covered in the book are the symmetrical arrangements of viruses; introduction to different families of animal viruses. Introduction to modern virology/N. J. Dimmock, A. J. Easton, K. N. Leppard. – 6th ed. .. This book, now in its sixth edition, provides a rounded introduction to.
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Anderson, Pekka E. Bernard J. Poiesz, Garth D. It also demonstrates the principal aspects of virus particle structure. Some of the topics covered in the book are the symmetrical arrangements of viruses; introduction to different families of animal viruses; biochemistry of virus particles; the immunological properties and biological activities of viral gene products; description of enzymatic activities of viruses; and haemagglutination, cell fusion, and haemolysis of viruses.
The description and characteristics of viral antigens are covered. The identification and propagation of viruses in tissue and cell cultures are discussed. An in-depth analysis of the principles of virus replication is provided.
A study of the morphogenesis of virions is also presented.
A chapter is devoted to virus-induced changes of cell structures and functions. The book can provide useful information to virologists, microbiologists, students, and researchers. We are always looking for ways to improve customer experience on Elsevier.
We would like to ask you for a moment of your time to fill in a short questionnaire, at the end of your visit. If you decide to participate, a new browser tab will open so you can complete the survey after you have completed your visit to this website. Thanks in advance for your time. Skip to content. Different technologies such as Roche [ 6 ], Illumina [ 7 ], Ion Torrent [ 8 ] and more recently the fourth-generation sequencing methodologies popularly called single-cell sequencing, viz.
Oxford Nanopore [ 9 ] and Pacific Biosciences [ 10 ], are available for sequencing. Sample preparation and enrichment are the prerequisites for sequencing the viromes. Filtration and centrifugation on caesium chloride density gradient have proved to enrich the virus-like particles.
A strategy like depletion of host rRNAs is also known to increase the virus fraction and has been attributed to the discovery of several novel RNA viruses [ 11 ]. There exist several scenarios for sequencing viral genomes such as sequencing of individual strains or population [ 12 ].
Sequencing of individual genomes helps to catalogue the genes encoded in a particular strain and is a vital step for in-depth characterization studies.
CRISPR are found in archaea and bacteria that serve as an antiviral mechanism in which viral genomic sequences are integrated as CRISPR spacers into the host, thereby making it immune to viral infection [ 15 ].
Understanding complex dynamics of virus—host interactions in higher organisms using sequencing provides valuable insights into transmission between animal reservoirs [ 16 ]. Sequencing of 'Auxiliary metabolic genes', which are involved in processes like motility and transcriptional repression, enables to unravel the viral genes that influence host machinery in diverse ways [ 17 ].
Data assembly and annotations Output from NGS technologies results in gigabases of raw sequence data per experiment. Extensive computational analysis using a number of algorithms and applications is required to infer biological significance. Care should be taken in case of paired-end sequences to ensure that the reads trimmed based on the quality is reflected in both the forward and the reverse FASTQ files.
In case of multiplex sequencing data, an additional step of 'de-multiplexing' based on barcodes is mandatory.
Filtering of such data ensures that no error is propagated. Following preprocessing, reference-based mapping or de novo assembly of the processed reads can be carried out. Reference-mapping Alignment with a reference genome is a method of choice for most NGS experiments. Preprocessed reads when mapped to a well-annotated reference genome ensure transfer of annotations to the query genome in a hassle-free manner with statistical confidence, especially in indel-free regions.
Polymorphic regions can also be identified, which account for the isolate-specific variants that may be responsible for the observed phenotype.
The algorithms generally rely on indexing of either the query reads or the reference genome using suffix tree or hashing strategy [ 20 — 22 ]. Indexing the reference genome has been proved to be computationally advantageous and is widely preferred. Indexing is followed by gapped or ungapped alignment based on either Smith—Waterman [ 23 ] or Needleman—Wunsch dynamic programming approaches [ 24 ].
The quality of the reference alignment can be improved by using large inserts available in paired-end reads as compared to single-end reads wherein forward and reverse orientation of reads cannot be calculated. Downstream processing of aligned and assembled reads involves delineating the variant regions followed by annotation.
It is also important to remove polymerase chain reaction PCR artefacts before variant calling as the duplicated reads hamper its sensitivity. Discovery of Schmallemberg virus, a new member of genus Orthobunyavirus that causes foetal abnormalities in ruminants [ 25 ], is attributed to a reference-based assembly approach. Delineation of variant regions: All deviations from reference genome can be delineated as variants, which include SNPs and indels.
Variant regions contribute to the nucleotide diversity in virus populations and hence play a vital role in their evolution and dynamics. One of the main parameters indicative of nucleotide diversity is the comparison of synonymous to non-synonymous codon substitution. Synonymous mutations result in neutral substitution, which enable in maintaining the phenotype, as compared to non-synonymous substitutions, which lead to amino acid alteration and hence may affect phenotype.
It is interesting to note that the existence of overlapping reading frames in viruses often constrains synonymous substitutions. Hence, computation of the magnitude of synonymous and non-synonymous polymorphism within viral populations will provide a handle to assess the role of neutral evolution and genetic drift in viral evolution.
A more detailed discussion of the role of these substitution ratios in adaptive evolution of viruses is given in Section 4.
Tools like SNPgenie [ 26 ] and VirVarSeq [ 27 ] have been developed with a focus on calling SNPs from pooled viral samples by including codon information in an explicit manner and hence are more sensitive than traditional SNP callers [ 28 , 29 ].
De novo assembly Preprocessed reads are assembled using de novo approaches, when a closely related homologue is unavailable to serve as a reference. It should be mentioned that genome assembly is computationally challenging and also requires trained manpower.
Sequencing depth plays a major role in determining the quality of the assembly as does the length of the reads. Popularly used assemblers are based on de Bruijin graph approach in which reads are divided into subsequences called k-mers of length k [ 30 ]. The k-mers form the nodes of a graph, which are linked when a k-1mer is shared among them.
The overall process requires large amounts of computer memory RAM and specialized compute clusters. The steps involved in assembly process are: Based on Overlap—Layout—Consensus principle, information stored in scattered reads are used to make contiguous regions termed 'contigs', which are generally devoid of polymorphisms. Using insert information, 'contigs' are combined to form 'scaffolds'.
Gaps between contigs are usually filled with nucleotides Ns. Scaffolds in conjunction with synteny and geneorder information are used to build larger scaffolds.
Building a draft genome is an iterative process and involves parameter optimization, and it is advised that more than one type of assembler be used as each of them has been built for a definite purpose and has unique features. The final assembled genome is evaluated on the basis of N50 parameter. N50 is the median of assembled sequence lengths, in which longer sequences are given more weightage. Mis-assemblies due to wrong orientation of reads and low-complexity regions are, however, not accounted for in N50 parameter and tools like amosvalidate, which combines multiple validation procedures, are recommended [ 31 ].
One of the major limitations of de novo assembly using NGS data is its reporting of large proportion of incorrect recombinants. This arises mainly due to overlapping of short reads of varying quality and coverage, which in turn pave way for the introduction of spurious SNPs, ultimately resulting in artefacts in assembly. The in silico chimeras thus produced amplify diversity estimation and complicate true recombination detection.
Efforts are being made to overcome this issue using probabilistic method, which assumes that true SNPs are under selection pressure and hence co-occur within a haplotype as compared to random SNPs [ 32 ]. Novel approaches are also being introduced with special emphasis on viral metagenomic projects, viz. Hence, de novo assembly has tremendous scope in unravelling the vast virome that has been unaddressed previously and there exists need for development of more efficient assembly algorithms, which will make it more tractable for use by larger scientific community.
Genome databases and resources dedicated to viruses were developed subsequently [ 43 — 47 ]. Lists of useful databases, resources and analysis tools have also been compiled previously [ 13 , 48 ]. Most of these resources archive complete genome sequences, their annotations and derived data such as viral variations, multiple sequence alignments MSAs and phylogenetic trees, to name a few.
Some of the viral genome resources are briefly described below. It attempts to curate reference genome sequences and leverages on the knowledge of experts to annotate as well as to identify important viral sequences. The objective of the resource is to link textbook knowledge, fact sheets and images to the genomic and proteomic data with an objective to facilitate the study of viral diversity [ 50 ].
The database currently provides access to molecular data of viruses including complete genomes of 14 viral families. Analytical and visualization tools for metadata-driven statistical sequence analysis, data filtering, analytical workflows and utility of personal workbench are provided to the users. Such annotations will be highly useful in subsequent analysis and model building. The challenges of managing dedicated resources for viral genomes are relatively different as compared to the genomic databases of model and other organisms.
The pace of sequencing and the quantum of genomic data being generated are affecting identification of reference genomes and annotations of genomes of strains and isolates. Additionally, to study the spatio-temporal evolution and to model the viral populations, it is desirable to tag metadata such as the place and date of isolation of viruses with the corresponding genomic entries.
While understanding the sequence—structure—function relationships, it has also resulted in the development of new areas of research such as phyloinformatics and immunoinformatics, which translates raw data into information. The information generated from these independent yet interlinked areas, when put together fits as pieces of jigsaw puzzle Figure 1 , leading to an improved understanding of the viral diseases and, thereby, the development of antiviral therapies.
Figure 1. Scope of research in virology enabled and augmented due to availability of NGS data.
Unravelling mutational landscapes in viral quasispecies Viral quasispecies are mutant swarms generated mainly by RNA viruses during replication, which is known to be error-prone due to the lack of proofreading activity of RNA-dependent RNA polymerase. The resulting mosaic is a dynamic distribution of non-identical but related replicons that cannot be detected using conventional sequencing approaches. Hence, quasispecies remained unexplored for a considerable time, even though the theoretical concept for quasispecies was put forth by Eigen in [ 55 ].
With the advent of NGS technologies, the generation of large genomic datasets became a reality. Due to the sequencing error issues, it was still tough to demarcate true genetic variations. Circular Sequencing CirSeq , a novel experimental approach that creates template of tandem repeats of circularized genomic RNA fragments has been developed by Andino's group [ 56 ].
CirSeq reduces the sequencing error drastically as the repeats get sequenced in a redundant manner for every genomic fragment. CirSeq was employed to study seven serial passages of Poliovirus replicated in HeLa cells. Mutation frequency was computed for every passage and their fitness was determined by mapping onto the 3D structure of proteins.
As expected, majority of the mutations detected were neutral substitutions, thus highlighting robustness as driving force for adaptation and evolution [ 56 ]. This study clearly delineates the viral mutations responsible for quasispecies structure and highlights the extent of genetic variation that can be maintained in a population.
Microevolution in an evolving quasispecies population is responsible for the sequence diversity in Porcine reproductive and respiratory syndrome virus PRRSV.
PRRSV is the causative agent of late-term reproductive failure in sows and respiratory distress in pigs and hence has large economic impact. Genomic complexity of PRRSV due to multiple circulating genotypes results in antigenic diversity, which, in turn, is responsible for lack of effective vaccine development [ 57 ].
Sanger sequencing has identified open reading frames ORF5 and ORF7 as the polymorphic regions of the virus genome, encoding major immunogenic epitopes. By analysing nucleotide substitutions over time followed by comparative genomics with non-pathogenic variants, the role of mutation and selection in preserving the pathogenesis or fitness of PRRSV was well documented in this study. Minority quasispecies refers to the memory genomes that were dominant at an earlier phase of quasispecies evolution and can play an important role in conferring drug resistance in viruses such as Human Immunodeficiency Virus type-1 HIV-1 and Influenza virus.
Minority quasispecies of drug-resistant viruses can rapidly re-emerge as major populations after the reintroduction of drug pressure.