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3295 KiB

Open AccessArticle

HIV-1 Mutation and Recombination Rates Are Different in Macrophages and T-cells

by Deborah Cromer, Timothy E. Schlub, Redmond P. Smyth, Andrew J. Grimm, Abha Chopra, Simon Mallal, Miles P. Davenport and Johnson Mak

Viruses 2016, 8(4), 118; https://doi.org/10.3390/v8040118 - 22 Apr 2016

Cited by 10 | Viewed by 7349

Abstract

High rates of mutation and recombination help human immunodeficiency virus (HIV) to evade the immune system and develop resistance to antiretroviral therapy. Macrophages and T-cells are the natural target cells of HIV-1 infection. A consensus has not been reached as to whether HIV [...] Read more.

High rates of mutation and recombination help human immunodeficiency virus (HIV) to evade the immune system and develop resistance to antiretroviral therapy. Macrophages and T-cells are the natural target cells of HIV-1 infection. A consensus has not been reached as to whether HIV replication results in differential recombination between primary T-cells and macrophages. Here, we used HIV with silent mutation markers along with next generation sequencing to compare the mutation and the recombination rates of HIV directly in T lymphocytes and macrophages. We observed a more than four-fold higher recombination rate of HIV in macrophages compared to T-cells (p < 0.001) and demonstrated that this difference is not due to different reliance on C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 5 (CCR5) co-receptors between T-cells and macrophages. We also found that the pattern of recombination across the HIV genome (hot and cold spots) remains constant between T-cells and macrophages despite a three-fold increase in the overall recombination rate. This indicates that the difference in rates is a general feature of HIV DNA synthesis during macrophage infection. In contrast to HIV recombination, we found that T-cells have a 30% higher mutation rate than macrophages (p < 0.001) and that the mutational profile is similar between these cell types. Unexpectedly, we found no association between mutation and recombination in macrophages, in contrast to T-cells. Our data highlights some of the fundamental difference of HIV recombination and mutation amongst these two major target cells of infection. Understanding these differences will provide invaluable insights toward HIV evolution and how the virus evades immune surveillance and anti-retroviral therapeutics. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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2186 KiB

Open AccessArticle

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region

by Enrico Lavezzo, Giulia Masi, Stefano Toppo, Elisa Franchin, Valentina Gazzola, Alessandro Sinigaglia, Serena Masiero, Marta Trevisan, Silvana Pagni, Giorgio Palù and Luisa Barzon

Viruses 2016, 8(3), 79; https://doi.org/10.3390/v8030079 - 14 Mar 2016

Cited by 18 | Viewed by 7332

Abstract

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity [...] Read more.

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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5921 KiB

Open AccessArticle

Characterization of Viral Communities of Biting Midges and Identification of Novel Thogotovirus Species and Rhabdovirus Genus

by Sarah Temmam, Sonia Monteil-Bouchard, Catherine Robert, Jean-Pierre Baudoin, Masse Sambou, Maxence Aubadie-Ladrix, Noémie Labas, Didier Raoult, Oleg Mediannikov and Christelle Desnues

Viruses 2016, 8(3), 77; https://doi.org/10.3390/v8030077 - 11 Mar 2016

Cited by 34 | Viewed by 8610

Abstract

More than two thirds of emerging viruses are of zoonotic origin, and among them RNA viruses represent the majority. Ceratopogonidae (genus Culicoides) are well-known vectors of several viruses responsible for epizooties (bluetongue, epizootic haemorrhagic disease, etc.). They are also vectors of [...] Read more.

More than two thirds of emerging viruses are of zoonotic origin, and among them RNA viruses represent the majority. Ceratopogonidae (genus Culicoides) are well-known vectors of several viruses responsible for epizooties (bluetongue, epizootic haemorrhagic disease, etc.). They are also vectors of the only known virus infecting humans: the Oropouche virus. Female midges usually feed on a variety of hosts, leading to possible transmission of emerging viruses from animals to humans. In this context, we report here the analysis of RNA viral communities of Senegalese biting midges using next-generation sequencing techniques as a preliminary step toward the identification of potential viral biohazards. Sequencing of the RNA virome of three pools of Culicoides revealed the presence of a significant diversity of viruses infecting plants, insects and mammals. Several novel viruses were detected, including a novel Thogotovirus species, related but genetically distant from previously described tick-borne thogotoviruses. Novel rhabdoviruses were also detected, possibly constituting a novel Rhabdoviridae genus, and putatively restricted to insects. Sequences related to the major viruses transmitted by Culicoides, i.e., African horse sickness, bluetongue and epizootic haemorrhagic disease viruses were also detected. This study highlights the interest in monitoring the emergence and circulation of zoonoses and epizooties using their arthropod vectors. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessCommunication

Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

by Hironobu Yanagisawa, Reiko Tomita, Koji Katsu, Takuya Uehara, Go Atsumi, Chika Tateda, Kappei Kobayashi and Ken-Taro Sekine

Viruses 2016, 8(3), 70; https://doi.org/10.3390/v8030070 - 7 Mar 2016

Cited by 15 | Viewed by 7650

Abstract

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of [...] Read more.

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Identification of Known and Novel Recurrent Viral Sequences in Data from Multiple Patients and Multiple Cancers

by Jens Friis-Nielsen, Kristín Rós Kjartansdóttir, Sarah Mollerup, Maria Asplund, Tobias Mourier, Randi Holm Jensen, Thomas Arn Hansen, Alba Rey-Iglesia, Stine Raith Richter, Ida Broman Nielsen, David E. Alquezar-Planas, Pernille V. S. Olsen, Lasse Vinner, Helena Fridholm, Lars Peter Nielsen, Eske Willerslev, Thomas Sicheritz-Pontén, Ole Lund, Anders Johannes Hansen, Jose M. G. Izarzugaza and Søren Brunak Show full author list Hide full author list

Viruses 2016, 8(2), 53; https://doi.org/10.3390/v8020053 - 19 Feb 2016

Cited by 10 | Viewed by 8282

Abstract

Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We [...] Read more.

Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. We applied the workflow to 686 sequencing libraries from 252 cancer samples of different cancer and tissue types, 32 non-template controls, and 24 test samples. Recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. We provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. Unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Metagenomic Analysis of Virioplankton of the Subtropical Jiulong River Estuary, China

by Lanlan Cai, Rui Zhang, Ying He, Xiaoyuan Feng and Nianzhi Jiao

Viruses 2016, 8(2), 35; https://doi.org/10.3390/v8020035 - 2 Feb 2016

Cited by 42 | Viewed by 8279

Abstract

Viruses are the most abundant biological entities in the oceans, and encompass a significant reservoir of genetic diversity. However, little is known about their biodiversity in estuary environments, which represent a highly dynamic and potentially more diverse habitat. Here, we report a metagenomic [...] Read more.

Viruses are the most abundant biological entities in the oceans, and encompass a significant reservoir of genetic diversity. However, little is known about their biodiversity in estuary environments, which represent a highly dynamic and potentially more diverse habitat. Here, we report a metagenomic analysis of the dsDNA viral community from the Jiulong River Estuary (JRE), China, and provide a comparative analysis with other closely related environments. The results showed that the majority of JRE virome did not show any significant similarity to the database. For the major viral group (Caudovirales) detected in the sample, Podoviridae (44.88%) were the most abundant family, followed by Siphoviridae (32.98%) and Myoviridae (17.32%). The two most abundant viruses identified in the virome were phages HTVC010P and HMO-2011, which infect bacteria belonging to marine SAR11 and SAR116 clades, respectively. Two contigs larger than 20 kb, which show similar overall genome architectures to Celeribacter phage P12053L and Thalosomonas phage BA3, respectively, were generated during assembly. Comparative analysis showed that the JRE virome was more similar to marine viromes than to freshwater viromes, and shared a relative coarse-grain genetic overlap (averaging 14.14% ± 1.68%) with other coastal viromes. Our study indicated that the diversity and community structure of the virioplankton found in JRE were mainly affected by marine waters, with less influence from freshwater discharge. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

HPV Population Profiling in Healthy Men by Next-Generation Deep Sequencing Coupled with HPV-QUEST

by Li Yin, Jin Yao, Kaifen Chang, Brent P. Gardner, Fahong Yu, Anna R. Giuliano and Maureen M. Goodenow

Viruses 2016, 8(2), 28; https://doi.org/10.3390/v8020028 - 25 Jan 2016

Cited by 8 | Viewed by 8628

Abstract

Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and [...] Read more.

Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and a bioinformatic pipeline, was established and applied to survey the diversity of HPV genotypes among a subset of healthy men from the HPV in Men (HIM) Multinational Study. Twenty-one HPV genotypes (12 high-risk and 9 low-risk) were detected in the genital area from 18 asymptomatic individuals. A single HPV type, either HPV16, HPV6b or HPV83, was detected in 7 individuals, while coinfection by 2 to 5 high-risk and/or low-risk genotypes was identified in the other 11 participants. In two individuals studied for over one year, HPV16 persisted, while fluctuations of coinfecting genotypes occurred. HPV L1 regions were generally identical between query and reference sequences, although nonsynonymous and synonymous nucleotide polymorphisms of HPV16, 18, 31, 35h, 59, 70, 73, cand85, 6b, 62, 81, 83, cand89 or JEB2 L1 genotypes, mostly unidentified by linear array, were evident. Deep sequencing coupled with HPV-QUEST provides efficient and unambiguous classification of HPV genotypes in multiple-type HPV infection in host ecosystems. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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1436 KiB

Open AccessArticle

Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection

by Joshua Tweedy, Maria Alexandra Spyrou, Max Pearson, Dirk Lassner, Uwe Kuhl and Ursula A. Gompels

Viruses 2016, 8(1), 19; https://doi.org/10.3390/v8010019 - 15 Jan 2016

Cited by 38 | Viewed by 7454

Abstract

Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated “CiHHV-6A/B”. These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent [...] Read more.

Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated “CiHHV-6A/B”. These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing

by Bram Vrancken, Nídia Sequeira Trovão, Guy Baele, Eric Van Wijngaerden, Anne-Mieke Vandamme, Kristel Van Laethem and Philippe Lemey

Viruses 2016, 8(1), 12; https://doi.org/10.3390/v8010012 - 7 Jan 2016

Cited by 14 | Viewed by 6604

Abstract

Genetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely [...] Read more.

Genetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure—from nucleic acid extraction to sequencing—should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

by Danielle P. Porter, Martin Daeumer, Alexander Thielen, Silvia Chang, Ross Martin, Cal Cohen, Michael D. Miller and Kirsten L. White

Viruses 2015, 7(12), 6360-6370; https://doi.org/10.3390/v7122943 - 7 Dec 2015

Cited by 20 | Viewed by 6222

Abstract

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared [...] Read more.

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

An Evolutionarily Young Polar Bear (Ursus maritimus) Endogenous Retrovirus Identified from Next Generation Sequence Data

by Kyriakos Tsangaras, Jens Mayer, David E. Alquezar-Planas and Alex D. Greenwood

Viruses 2015, 7(11), 6089-6107; https://doi.org/10.3390/v7112927 - 24 Nov 2015

Cited by 7 | Viewed by 7945

Abstract

Transcriptome analysis of polar bear (Ursus maritimus) tissues identified sequences with similarity to Porcine Endogenous Retroviruses (PERV). Based on these sequences, four proviral copies and 15 solo long terminal repeats (LTRs) of a newly described endogenous retrovirus were characterized from the [...] Read more.

Transcriptome analysis of polar bear (Ursus maritimus) tissues identified sequences with similarity to Porcine Endogenous Retroviruses (PERV). Based on these sequences, four proviral copies and 15 solo long terminal repeats (LTRs) of a newly described endogenous retrovirus were characterized from the polar bear draft genome sequence. Closely related sequences were identified by PCR analysis of brown bear (Ursus arctos) and black bear (Ursus americanus) but were absent in non-Ursinae bear species. The virus was therefore designated UrsusERV. Two distinct groups of LTRs were observed including a recombinant ERV that contained one LTR belonging to each group indicating that genomic invasions by at least two UrsusERV variants have recently occurred. Age estimates based on proviral LTR divergence and conservation of integration sites among ursids suggest the viral group is only a few million years old. The youngest provirus was polar bear specific, had intact open reading frames (ORFs) and could potentially encode functional proteins. Phylogenetic analyses of UrsusERV consensus protein sequences suggest that it is part of a pig, gibbon and koala retrovirus clade. The young age estimates and lineage specificity of the virus suggests UrsusERV is a recent cross species transmission from an unknown reservoir and places the viral group among the youngest of ERVs identified in mammals. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Genomic Mining Reveals Deep Evolutionary Relationships between Bornaviruses and Bats

by Jie Cui and Lin-Fa Wang

Viruses 2015, 7(11), 5792-5800; https://doi.org/10.3390/v7112906 - 10 Nov 2015

Cited by 11 | Viewed by 6670

Abstract

Bats globally harbor viruses in order Mononegavirales, such as lyssaviruses and henipaviruses; however, little is known about their relationships with bornaviruses. Previous studies showed that viral fossils of bornaviral origin are embedded in the genomes of several mammalian species such as primates, indicative [...] Read more.

Bats globally harbor viruses in order Mononegavirales, such as lyssaviruses and henipaviruses; however, little is known about their relationships with bornaviruses. Previous studies showed that viral fossils of bornaviral origin are embedded in the genomes of several mammalian species such as primates, indicative of an ancient origin of exogenous bornaviruses. In this study, we mined the available 10 bat genomes and recreated a clear evolutionary relationship of endogenous bornaviral elements and bats. Comparative genomics showed that endogenization of bornaviral elements frequently occurred in vesper bats, harboring EBLLs (endogenous bornavirus-like L elements) in their genomes. Molecular dating uncovered a continuous bornavirus-bat interaction spanning 70 million years. We conclude that better understanding of modern exogenous bornaviral circulation in bat populations is warranted. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessArticle

Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

by Aurélien Chateigner, Annie Bézier, Carole Labrousse, Davy Jiolle, Valérie Barbe and Elisabeth A. Herniou

Viruses 2015, 7(7), 3625-3646; https://doi.org/10.3390/v7072788 - 7 Jul 2015

Cited by 53 | Viewed by 10141

Abstract

Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a [...] Read more.

Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Review

Jump to: Research

656 KiB

Open AccessReview

Single-Cell Genomics for Virology

by Angela Ciuffi, Sylvie Rato and Amalio Telenti

Viruses 2016, 8(5), 123; https://doi.org/10.3390/v8050123 - 4 May 2016

Cited by 32 | Viewed by 8552

Abstract

Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore [...] Read more.

Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessReview

Next-Generation Sequencing in the Understanding of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Biology

by Roxanne Strahan, Timsy Uppal and Subhash C. Verma

Viruses 2016, 8(4), 92; https://doi.org/10.3390/v8040092 - 31 Mar 2016

Cited by 7 | Viewed by 10017

Abstract

Non-Sanger-based novel nucleic acid sequencing techniques, referred to as Next-Generation Sequencing (NGS), provide a rapid, reliable, high-throughput, and massively parallel sequencing methodology that has improved our understanding of human cancers and cancer-related viruses. NGS has become a quintessential research tool for more effective [...] Read more.

Non-Sanger-based novel nucleic acid sequencing techniques, referred to as Next-Generation Sequencing (NGS), provide a rapid, reliable, high-throughput, and massively parallel sequencing methodology that has improved our understanding of human cancers and cancer-related viruses. NGS has become a quintessential research tool for more effective characterization of complex viral and host genomes through its ever-expanding repertoire, which consists of whole-genome sequencing, whole-transcriptome sequencing, and whole-epigenome sequencing. These new NGS platforms provide a comprehensive and systematic genome-wide analysis of genomic sequences and a full transcriptional profile at a single nucleotide resolution. When combined, these techniques help unlock the function of novel genes and the related pathways that contribute to the overall viral pathogenesis. Ongoing research in the field of virology endeavors to identify the role of various underlying mechanisms that control the regulation of the herpesvirus biphasic lifecycle in order to discover potential therapeutic targets and treatment strategies. In this review, we have complied the most recent findings about the application of NGS in Kaposi’s sarcoma-associated herpesvirus (KSHV) biology, including identification of novel genomic features and whole-genome KSHV diversities, global gene regulatory network profiling for intricate transcriptome analyses, and surveying of epigenetic marks (DNA methylation, modified histones, and chromatin remodelers) during de novo, latent, and productive KSHV infections. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessReview

From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes

by Hin Kwok and Alan Kwok Shing Chiang

Viruses 2016, 8(3), 60; https://doi.org/10.3390/v8030060 - 24 Feb 2016

Cited by 17 | Viewed by 8674

Abstract

Genomic sequences of Epstein–Barr virus (EBV) have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the [...] Read more.

Genomic sequences of Epstein–Barr virus (EBV) have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS) and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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Open AccessReview

Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology

by Ge Gao and David I. Smith

Viruses 2015, 7(8), 4507-4528; https://doi.org/10.3390/v7082831 - 7 Aug 2015

Cited by 5 | Viewed by 8122

Abstract

DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a [...] Read more.

DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients. Full article

(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)

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