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Asset Tag: Viral

April 21, 2020

Combining Viral Genetics and Statistical Modeling to Improve HIV-1 Time-of-infection Estimation towards Enhanced Vaccine Efficacy Assessment.

Knowledge of the time of HIV-1 infection and the multiplicity of viruses that establish HIV-1 infection is crucial for the in-depth analysis of clinical prevention efficacy trial outcomes. Better estimation methods would improve the ability to characterize immunological and genetic sequence correlates of efficacy within preventive efficacy trials of HIV-1 vaccines and monoclonal antibodies. We developed new methods for infection timing and multiplicity estimation using maximum likelihood estimators that shift and scale (calibrate) estimates by fitting true infection times and founder virus multiplicities to a linear regression model with independent variables defined by data on HIV-1 sequences, viral load, diagnostics, and sequence alignment statistics. Using Poisson models of measured mutation counts and phylogenetic trees, we analyzed longitudinal HIV-1 sequence data together with diagnostic and viral load data from the RV217 and CAPRISA 002 acute HIV-1 infection cohort studies. We used leave-one-out cross validation to evaluate the prediction error of these calibrated estimators versus that of existing estimators and found that both infection time and founder multiplicity can be estimated with improved accuracy and precision by calibration. Calibration considerably improved all estimators of time since HIV-1 infection, in terms of reducing bias to near zero and reducing root mean squared error (RMSE) to 5-10 days for sequences collected 1-2 months after infection. The calibration of multiplicity assessments yielded strong improvements with accurate predictions (ROC-AUC above 0.85) in all cases. These results have not yet been validated on external data, and the best-fitting models are likely to be less robust than simpler models to variation in sequencing conditions. For all evaluated models, these results demonstrate the value of calibration for improved estimation of founder multiplicity and of time since HIV-1 infection.

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April 21, 2020

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

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April 21, 2020

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt’s lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3′ ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62?pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3′ rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV+) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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April 21, 2020

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field.

We present a new method, CIDER-Seq (Circular DNA Enrichment sequencing) for the unbiased enrichment and long-read sequencing of viral-sized circular DNA molecules. We used CIDER-Seq to produce single-read full-length virus genomes for the first time. CIDER-Seq combines PCR-free virus enrichment with Single Molecule Real Time sequencing and a new sequence de-concatenation algorithm. We apply our technique to produce >1200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral double-stranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our method and its application in an economically important crop plant opens new possibilities in periodic virus sequence surveillance and accurate profiling of diverse circular DNA elements.

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April 21, 2020

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called ‘amplicon denoising’, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection.

HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFN? responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted dendritic cell (DC) maturation and CD4 T cell trans-infection perhaps explaining why AE are predominant in CHI. Taken together, our data suggests that the emergence of AE-specific CD8 T cell responses in CHI confers a selective advantage to the virus by promoting DC-mediated CD4 T cell trans-infection.

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April 21, 2020

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

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April 21, 2020

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads-the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process.

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April 21, 2020

Long-range PCR and high-throughput sequencing of Ostreid herpesvirus 1 indicate high genetic diversity and complex evolution process.

Ostreid herpesvirus 1 (OsHV-1) is an important pathogen associated with mass mortalities of cultivated marine mollusks worldwide. Since no cell line allows OsHV-1 replication in vitro, it is difficult to isolate enough high-purity viral DNA for High-Throughput Sequencing (HTS). We developed an efficient approach for the enrichment of OsHV-1 DNA for HTS with long-range PCR. Twenty-three primer pairs were designed to cover 99.3% of the reference genome, and their performances were examined on ten OsHV-1 infected samples. Amplicon mixtures from six successfully amplified samples were sequenced with Illumina platform, and one of them (ZK0118) was also sequenced with the PacBio platform. PacBio reads were assembled into 2 scaffolds compared to 9-68 scaffolds assembled from the Illumina reads. Genomic comparison confirmed high genetic diversity among OsHV-1 variants. Phylogenetic analysis revealed that OsHV-1 evolution was mainly impacted by its host species rather than spatial segregation. Copyright © 2018 Elsevier Inc. All rights reserved.

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April 21, 2020

SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection.

Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.

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April 21, 2020

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Long-Read Sequencing – A Powerful Tool in Viral Transcriptome Research.

Long-read sequencing (LRS) has become increasingly popular due to its strengths in de novo assembly and in resolving complex DNA regions as well as in determining full-length RNA molecules. Two important LRS technologies have been developed during the past few years, including single-molecule, real-time sequencing by Pacific Biosciences, and nanopore sequencing by Oxford Nanopore Technologies. Although current LRS methods produce lower coverage, and are more error prone than short-read sequencing, these methods continue to be superior in identifying transcript isoforms including multispliced RNAs and transcript-length variants as well as overlapping transcripts and alternative polycistronic RNA molecules. Viruses have small, compact genomes and therefore these organisms are ideal subjects for transcriptome analysis with the relatively low-throughput LRS techniques. Recent LRS studies have multiplied the number of previously known transcripts and have revealed complex networks of transcriptional overlaps in the examined viruses.Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

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April 21, 2020

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, ?HV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome. Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Current advances in HIV vaccine preclinical studies using Macaque models.

The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model’s true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020

Analyses of four new Caulobacter Phicbkviruses indicate independent lineages.

Bacteriophages with genomes larger than 200 kbp are considered giant phages, and the giant Phicbkviruses are the most frequently isolated Caulobacter crescentus phages. In this study, we compare six bacteriophage genomes that differ from the genomes of the majority of Phicbkviruses. Four of these genomes are much larger than those of the rest of the Phicbkviruses, with genome sizes that are more than 250 kbp. A comparison of 16 Phicbkvirus genomes identified a ‘core genome’ of 69 genes that is present in all of these Phicbkvirus genomes, as well as shared accessory genes and genes that are unique for each phage. Most of the core genes are clustered into the regions coding for structural proteins or those involved in DNA replication. A phylogenetic analysis indicated that these 16 CaulobacterPhicbkvirus genomes are related, but they represent four distinct branches of the Phicbkvirus genomic tree with distantly related branches sharing little nucleotide homology. In contrast, pairwise comparisons within each branch of the phylogenetic tree showed that more than 80?% of the entire genome is shared among phages within a group. This conservation of the genomes within each branch indicates that horizontal gene transfer events between the groups are rare. Therefore, the Phicbkvirus genus consists of at least four different phylogenetic branches that are evolving independently from one another. One of these branches contains a 27-gene inversion relative to the other three branches. Also, an analysis of the tRNA genes showed that they are relatively mobile within the Phicbkvirus genus.

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