Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis).

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Human Cytomegalovirus RNA2.7 Is Required for Upregulating Multiple Cellular Genes To Promote Cell Motility and Viral Spread Late in Lytic Infection.

Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

Multiple-Strain Infections of Human Cytomegalovirus With High Genomic Diversity Are Common in Breast Milk From Human Immunodeficiency Virus-Infected Women in Zambia.

BACKGROUND: In developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, where multiple-strain infection appears linked to disease severity. The situation is less documented in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in human immunodeficiency virus (HIV)-infected women. We investigated HCMV strain diversity. METHODS: High-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-infected and 7 HIV-negative) at 4-16 weeks postpartum, then analyzed by genome assembly and novel motif-based genotyping in 12 hypervariable HCMV genes. RESULTS: Among the 20 samples from 14 donors (13 HIV-infected and one HIV-negative) who yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to 5 strains per person were apparent in 9 HIV-infected women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus entry and immunomodulation, but not between genes more distant from each other. CONCLUSIONS: Breast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.

An analysis of codon bias in six red yeast species.

Red yeasts, primarily species of Rhodotorula, Sporobolomyces, and other genera of Pucciniomycotina, are traditionally considered proficient systems for lipid and terpene production, and only recently have also gained consideration for the production of a wider range of molecules of biotechnological potential. Improvements of transgene delivery protocols and regulated gene expression systems have been proposed, but a dearth of information on compositional and/or structural features of genes has prevented transgene sequence optimization efforts for high expression levels. Here, the codon compositional features of genes in six red yeast species were characterized, and the impact that evolutionary forces may have played in shaping this compositional bias was dissected by using several computational approaches. Results obtained are compatible with the hypothesis that mutational bias, although playing a significant role, cannot alone explain synonymous codon usage bias of genes. Nevertheless, several lines of evidences indicated a role for translational selection in driving the synonymous codons that allow high expression efficiency. These optimal synonymous codons are identified for each of the six species analyzed. Moreover, the presence of intragenic patterns of codon usage, which are thought to facilitate polyribosome formation, was highlighted. The information presented should be taken into consideration for transgene design for optimal expression in red yeast species.

CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

Survey of Brevibacillus laterosporus insecticidal protein genes and virulence factors.

The pathogenic action of the bacterium Brevibacillus laterosporus against invertebrates involves a toxin-mediated mechanism. Several studies, conducted with specific strains against diverse targets, suggested the implication of different toxins. Recent genome sequencing and annotation of some insecticidal strains revealed several putative virulence factors highly conserved in this species. After determining the pathogenicity of strain UNISS 18 against different Lepidopteran and Dipteran larvae, in this study we have investigated the actual expression of genes encoding for enzymes (i.e., chitinases, proteases), toxins, and other virulence factors, either in vitro and in vivo at the transcriptional level. Selected genes encode for two chitinases, a collagenase-like protease, a GlcNAc-binding protein, two protective antigen proteins, a bacillolysin, a thermophilic serine proteinase, two spore surface proteins, an insecticidal toxin homologous to Cry75Aa. All target genes were well expressed in pure bacterial cultures with significant differences between bacterial growth phases. Their expression level was generally enhanced in the bacterial population developing in the insect body cavity, compared with pure culture. The expression of certain genes increased substantially over time after insect inoculation. These results support a complex mechanism of action leveraging a variety of available virulence factors, and can also explain the ability of this bacterial species to act against diverse invertebrate targets.

The dynamic loss and gain of introns during the evolution of the Brassicaceae.

Sequence comparison allows the detailed analysis of evolution at the nucleotide and amino acid levels, but much less information is known about the structural evolution of genes, i.e. how the number, length and distribution of introns change over time. We constructed a parsimonious model for the evolutionary rate of intron loss (IL) and intron gain (IG) within the Brassicaceae and found that IL/IG has been highly dynamic, with substantial differences between and even within lineages. The divergence of the Brassicaceae lineages I and II marked a dramatic change in the IL rate, with the common ancestor of lineage I losing introns three times more rapidly than the common ancestor of lineage II. Our data also indicate a subsequent declining trend in the rate of IL, although in Arabidopsis thaliana introns continue to be lost at approximately the ancestral rate. Variations in the rate of IL/IG within lineage II have been even more remarkable. Brassica rapa appears to have lost introns approximately 15 times more rapidly than the common ancestor of B. rapa and Schenkiella parvula, and approximately 25 times more rapidly than its sister species Eutrema salsugineum. Microhomology was detected at the splice sites of several dynamic introns suggesting that the non-homologous end-joining and double-strand break repair is a common pathway underlying IL/IG in these species. We also detected molecular signatures typical of mRNA-mediated IL, but only in B. rapa.

Unusual Stepwise Protonation and J-Aggregation of meso-Tetrakis(N-methylpyridinium-4-yl)porphine on Binding Poly(sodium vinylsulfonate).

The binding of a tetracationic porphyrin to a highly charged polymer like poly(sodium vinylsulfonate) has been investigated over a wide pH range and under various experimental conditions. We present evidence that, depending on the pH, the high electrostatic field exerted by the polymer stabilizes the diprotonated form of the free base porphyrin at unusual pH values or otherwise causes the formation of H-type aggregates. In particular, at a low polymer concentration, lowering the pH at first allows the formation of the diacid species then it determines its reorganization in close-packed J-type aggregates. The employment of various metallo-derivatives of the title porphyrin enables a better insight into the nature of all the detected species.

Direct Nanopore Sequencing of Human Cytomegalovirus Genomes from High-Viral-Load Clinical Samples.

Nanopore sequencing is becoming increasingly commonplace in clinical settings, particularly for diagnostic assessments and outbreak investigations, due to its portability, low cost, and ability to operate in near real-time. Although high sequencing error rates initially hampered the wider implementation of this technology, improvements have been made continually with each iteration of the sequencing hardware and base-calling software. Here, we assess the feasibility of using nanopore sequencing to determine the complete genomes of human cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a hybrid bioinformatic approach that involved assembling the reads de novo, improving the consensus sequence by aligning reads to the best-matching genome from a collated set of published sequences, and polishing the improved consensus sequence. The final genomes from a urine sample and a lung sample, the former with an HCMV to human DNA load approximately 50 times greater than the latter, achieved 99.97 and 99.93% identity, respectively, to the benchmark genomes obtained independently by Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of determining HCMV genomes directly from high-viral-load clinical samples with a high accuracy.

Complexity of Human Cytomegalovirus Infection in South African HIV-Exposed Infants with Pneumonia.

Human cytomegalovirus (HCMV) can cause significant end-organ diseases such as pneumonia in HIV-exposed infants. Complex viral factors may influence pathogenesis including: a large genome with a sizeable coding capacity, numerous gene regions of hypervariability, multiple-strain infections, and tissue compartmentalization of strains. We used a whole genome sequencing approach to assess the complexity of infection by comparing high-throughput sequencing data obtained from respiratory and blood specimens of HIV-exposed infants with severe HCMV pneumonia with those of lung transplant recipients and patients with hematological disorders. There were significantly more specimens from HIV-exposed infants showing multiple HCMV strain infection. Some genotypes, such as UL73 G4B and UL74 G4, were significantly more prevalent in HIV-exposed infants with severe HCMV pneumonia. Some genotypes were predominant in the respiratory specimens of several patients. However, the predominance was not statistically significant, precluding firm conclusions on anatomical compartmentalization in the lung.

Identifying high-confidence variants in human cytomegalovirus genomes sequenced from clinical samples.

Understanding the intrahost evolution of viral populations has implications in pathogenesis, diagnosis, and treatment and has recently made impressive advances from developments in high-throughput sequencing. However, the underlying analyses are very sensitive to sources of bias, error, and artefact in the data, and it is important that these are addressed adequately if robust conclusions are to be drawn. The key factors include (1) determining the number of viral strains present in the sample analysed; (2) monitoring the extent to which the data represent these strains and assessing the quality of these data; (3) dealing with the effects of cross-contamination; and (4) ensuring that the results are reproducible. We investigated these factors by generating sequence datasets, including biological and technical replicates, directly from clinical samples obtained from a small cohort of patients who had been infected congenitally with the herpesvirus human cytomegalovirus, with the aim of developing a strategy for identifying high-confidence intrahost variants. We found that such variants were few in number and typically present in low proportions and concluded that human cytomegalovirus exhibits a very low level of intrahost variability. In addition to clarifying the situation regarding human cytomegalovirus, our strategy has wider applicability to understanding the intrahost variability of other viruses.

GRACy: A tool for analysing human cytomegalovirus sequence data.

Modern DNA sequencing has instituted a new era in human cytomegalovirus (HCMV) genomics. A key development has been the ability to determine the genome sequences of HCMV strains directly from clinical material. This involves the application of complex and often non-standardized bioinformatics approaches to analysing data of variable quality in a process that requires substantial manual intervention. To relieve this bottleneck, we have developed GRACy (Genome Reconstruction and Annotation of Cytomegalovirus), an easy-to-use toolkit for analysing HCMV sequence data. GRACy automates and integrates modules for read filtering, genotyping, genome assembly, genome annotation, variant analysis, and data submission. These modules were tested extensively on simulated and experimental data and outperformed generic approaches. GRACy is written in Python and is embedded in a graphical user interface with all required dependencies installed by a single command. It runs on the Linux operating system and is designed to allow the future implementation of a cross-platform version. GRACy is distributed under a GPL 3.0 license and is freely available at https://bioinformatics.cvr.ac.uk/software/ with the manual and a test dataset.

LoReTTA, a user-friendly tool for assembling viral genomes from PacBio sequence data.

Long-read, single-molecule DNA sequencing technologies have triggered a revolution in genomics by enabling the determination of large, reference-quality genomes in ways that overcome some of the limitations of short-read sequencing. However, the greater length and higher error rate of the reads generated on long-read platforms make the tools used for assembling short reads unsuitable for use in data assembly and motivate the development of new approaches. We present LoReTTA (Long Read Template-Targeted Assembler), a tool designed for performing de novo assembly of long reads generated from viral genomes on the PacBio platform. LoReTTA exploits a reference genome to guide the assembly process, an approach that has been successful with short reads. The tool was designed to deal with reads originating from viral genomes, which feature high genetic variability, possible multiple isoforms, and the dominant presence of additional organisms in clinical or environmental samples. LoReTTA was tested on a range of simulated and experimental datasets and outperformed established long-read assemblers in terms of assembly contiguity and accuracy. The software runs under the Linux operating system, is designed for easy adaptation to alternative systems, and features an automatic installation pipeline that takes care of the required dependencies. A command-line version and a user-friendly graphical interface version are available under a GPLv3 license at https://bioinformatics.cvr.ac.uk/software/ with the manual and a test dataset.

Genome Sequence of Human Cytomegalovirus Ig-KG-H2, a Variant of Strain KG Propagated in the Presence of Neutralizing Antibodies.

Human cytomegalovirus shed in infant urine was isolated and serially passaged in fibroblasts in the presence or absence of neutralizing antibodies. Comparison of the genome sequences of representative viruses Ig-KG-H2 (passed with antibody) and ϕ-KG-B5 (passed without antibody) revealed the presence of several mutations in each virus.

Whole-Genome Approach to Assessing Human Cytomegalovirus Dynamics in Transplant Patients Undergoing Antiviral Therapy.

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, n = 9, and allogeneic hematopoietic stem cell, n = 2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations (n = 6), two (A594V and L595S) at high (100%) and four (V715M, V781I, A809V, and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high-throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy.

corseq: fast and efficient identification of favoured codons from next generation sequencing reads.

BACKGROUND: Optimization of transgene expression can be achieved by designing coding sequences with the synonymous codon usage of genes which are highly expressed in the host organism. The identification of the so-called "favoured codons" generally requires the access to either the genome or the coding sequences and the availability of expression data. RESULTS: Here we describe corseq, a fast and reliable software for detecting the favoured codons directly from RNAseq data without prior knowledge of genomic sequence or gene annotation. The presented tool allows the inference of codons that are preferentially used in highly expressed genes while estimating the transcripts abundance by a new kmer based approach. corseq is implemented in Python and runs under any operating system. The software requires the Biopython 1.65 library (or later versions) and is available under the 'GNU General Public License version 3' at the project webpage https://sourceforge.net/projects/corseq/files. CONCLUSION: corseq represents a faster and easy-to-use alternative for the detection of favoured codons in non model organisms.

Identification of Pseudogenes in Brachypodium distachyon Chromosomes.

Pseudogenes are gene copies that have lost the capability to encode a functional protein. Based on their structure, pseudogenes are classified in two types. Processed pseudogenes arise by a process of retrotranscription from a spliced mRNA and subsequent integration into the genome. Nonprocessed (or duplicated) pseudogenes are generated by genomic duplication and subsequent mutations that disable their functionality so that they cannot longer encode a functional protein. Differently from processed pseudogenes, duplicated pseudogenes are expected to conserve the exon-intron structure of their functional paralogs.Here, we describe a computational pipeline for identifying pseudogenes of both types in B. distachyon chromosomes. Our pipeline (1) identifies pseudogenes based on tBLASTn searches of B. distachyon proteins against the noncoding genomic complement of the same species, (2) identifies the most homologous pseudogenes functionally paralogous as the pseudogene paternal locus, (3) uses the intron-exon structure of paternal genes to distinguish between pseudogene types.The pipeline is presented in its composing steps and tested on the Brachypodium distachyon Bd1 scaffold.

The Evolutionary Basis of Translational Accuracy in Plants.

Mistranslation errors compromise fitness by wasting resources on nonfunctional proteins. In order to reduce the cost of mistranslations, natural selection chooses the most accurately translated codons at sites that are particularly important for protein structure and function. We investigated the determinants underlying selection for translational accuracy in several species of plants belonging to three clades: Brassicaceae, Fabidae, and Poaceae. Although signatures of translational selection were found in genes from a wide range of species, the underlying factors varied in nature and intensity. Indeed, the degree of synonymous codon bias at evolutionarily conserved sites varied among plant clades while remaining uniform within each clade. This is unlikely to solely reflect the diversity of tRNA pools because there is little correlation between synonymous codon bias and tRNA abundance, so other factors must affect codon choice and translational accuracy in plant genes. Accordingly, synonymous codon choice at a given site was affected not only by the selection pressure at that site, but also its participation in protein domains or mRNA secondary structures. Although these effects were detected in all the species we analyzed, their impact on translation accuracy was distinct in evolutionarily distant plant clades. The domain effect was found to enhance translational accuracy in dicot and monocot genes with a high GC content, but to oppose the selection of more accurate codons in monocot genes with a low GC content.