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Immune recognition of viral genome-derived double-stranded RNA (dsRNA) molecules and their subsequent processing into small interfering RNAs (siRNAs) in plants, invertebrates, and mammals trigger specific antiviral immunity known as antiviral RNA interference (RNAi). Immune sensing of viral dsRNA is sequence-independent, and most regions of viral RNAs are targeted by virus-derived siRNAs which extensively overlap in sequence. Thus, the high mutation rates of viruses do not drive immune escape from antiviral RNAi, in contrast to other mechanisms involving specific virus recognition by host immune proteins such as antibodies and resistance (R) proteins in mammals and plants, respectively. Instead, viruses actively suppress antiviral RNAi at various key steps with a group of proteins known as viral suppressors of RNAi (VSRs). Some VSRs are so effective in virus counter-defense that potent inhibition of virus infection by antiviral RNAi is undetectable unless the cognate VSR is rendered nonexpressing or nonfunctional. Since viral proteins are often multifunctional, resistance phenotypes of antiviral RNAi are accurately defined by those infection defects of VSR-deletion mutant viruses that are efficiently rescued by host deficiency in antiviral RNAi. Here, we review and discuss in vivo infection defects of VSR-deficient RNA and DNA viruses resulting from the actions of host antiviral RNAi in model systems.

Subcellular organization is essential for life. Cells organize their functions into organelles to concentrate their machinery and supplies for optimal efficiency. Likewise, viruses organize their replication machinery into compartments or factories within their host cells for optimal replicative efficiency. In this review, we discuss how DNA viruses that infect both eukaryotic cells and bacteria assemble replication compartments for synthesis of progeny viral DNA and transcription of the viral genome. Eukaryotic DNA viruses assemble replication compartments in the nucleus of the host cell while DNA bacteriophages assemble compartments called phage nuclei in the bacterial cytoplasm. Thus, DNA viruses infecting host cells from different domains of life share common replication strategies.

Nudiviruses (Nudiviridae) are double-stranded DNA viruses with enveloped and rod-shaped virions. Several insect orders (e.g., Diptera, Lepidoptera, Coleoptera, Orthoptera) and aquatic crustaceans are susceptible to nudivirus infections, which can result in varied degrees of disease in all developmental host stages. Their pathogenicity endangers insect rearing and crustacean aquacultures, but has also proven effective in biocontrol against Oryctes rhinoceros infestations. This literature review aims to present all known nudivirus species and provide a comprehensive Nudiviridae phylogeny by including recently described nudiviral isolates, and discuss this phylogeny in comparison to current opinions and taxonomical propositions. Moreover, we aim to clarify biological, pathological and genomic differences or similarities between nudiviruses and related entomopathogenic viruses, including baculoviruses (Baculoviridae) and bracoviruses (Polydnaviridae). A phylogenetic analysis using 17 concatenated nudivirus core genes resulted in the expected structure with the genera Alphanudivirus and Betanudivirus, as well as the most recently recognized genera Gammanudivirus and Deltanudivirus. The hymenopteran Osmia cornuta nudivirus (OcNV) groups closest with the hymenopteran Fopius arisanus endogenous nudivirus (FaENV) and does not share a most common ancestor with the hymenopteran bracoviruses. Except for one node, all clades are highly supported. The proposition of a recent study to assign subgroups to the alphanudiviruses might be legitimate, but more hymenopteran and orthopteran nudiviruses, especially in bees and cricket, need to be identified to resolve this proposal. In addition, freshwater and marine nudiviruses might form taxonomic subgroups among gammanudiviruses as well, but more aquatic nudiviruses need to be identified and sequenced for better resolution. Furthermore, the search for nudiviruses in insects with (semi)aquatic life stages may aid in finding the missing l

BackgroundViruses are a significant player in many biosphere and human ecosystems, but most signals remain "hidden" in metagenomic/metatranscriptomic sequence datasets due to the lack of universal gene markers, database representatives, and insufficiently advanced identification tools.ResultsHere, we introduce VirSorter2, a DNA and RNA virus identification tool that leverages genome-informed database advances across a collection of customized automatic classifiers to improve the accuracy and range of virus sequence detection. When benchmarked against genomes from both isolated and uncultivated viruses, VirSorter2 uniquely performed consistently with high accuracy (F1-score > 0.8) across viral diversity, while all other tools under-detected viruses outside of the group most represented in reference databases (i.e., those in the order Caudovirales). Among the tools evaluated, VirSorter2 was also uniquely able to minimize errors associated with atypical cellular sequences including eukaryotic genomes and plasmids. Finally, as the virosphere exploration unravels novel viral sequences, VirSorter2's modular design makes it inherently able to expand to new types of viruses via the design of new classifiers to maintain maximal sensitivity and specificity.ConclusionWith multi-classifier and modular design, VirSorter2 demonstrates higher overall accuracy across major viral groups and will advance our knowledge of virus evolution, diversity, and virus-microbe interaction in various ecosystems. Source code of VirSorter2 is freely available ( https://bitbucket.org/MAVERICLab/virsorter2 ), and VirSorter2 is also available both on bioconda and as an iVirus app on CyVerse ( https://de.cyverse.org/de ). Video abstract.

Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80-100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

The concentration of atmospheric methane (CH4) continues to increase with microbial communities controlling soil-atmosphere fluxes. While there is substantial knowledge of the diversity and function of prokaryotes regulating CH4 production and consumption, their active interactions with viruses in soil have not been identified. Metagenomic sequencing of soil microbial communities enables identification of linkages between viruses and hosts. However, this does not determine if these represent current or historical interactions nor whether a virus or host are active. In this study, we identified active interactions between individual host and virus populations in situ by following the transfer of assimilated carbon. Using DNA stable-isotope probing combined with metagenomic analyses, we characterized CH4-fueled microbial networks in acidic and neutral pH soils, specifically primary and secondary utilizers, together with the recent transfer of CH4-derived carbon to viruses. A total of 63% of viral contigs from replicated soil incubations contained homologs of genes present in known methylotrophic bacteria. Genomic sequences of 13C-enriched viruses were represented in over one-third of spacers in CRISPR arrays of multiple closely related Methylocystis populations and revealed differences in their history of viral interaction. Viruses infecting nonmethanotrophic methylotrophs and heterotrophic predatory bacteria were also identified through the analysis of shared homologous genes, demonstrating that carbon is transferred to a diverse range of viruses associated with CH4-fueled microbial food networks.

Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.

Piwi-interacting RNAs (piRNAs) are a class of small RNAs primarily responsible for silencing transposons in the animal germ line. The ping-pong cycle, the posttranscriptional silencing branch of the piRNA pathway, relies on piRNAs produced from endogenous transposon remnants to direct cleavage of transposon RNA via association with Piwi-family Argonaute proteins. In some mosquito species and mosquito-derived cell lines expressing a functionally expanded group of Piwi-family Argonaute proteins, both RNA and DNA viruses are targeted by piRNAs in a manner thought to involve direct processing of exogenous viral RNA into piRNAs. Whether viruses are targeted by piRNAs in nonmosquito species is unknown. Partial integrations of DNA and nonretroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce piRNAs that are speculated to target cognate viruses through the ping-pong cycle. Here, we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent virus-derived piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results demonstrate that viruses can be targeted by piRNAs in a nonmosquito species independently of endogenous piRNAs.IMPORTANCE Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct antitransposon responses. These responses require endogenous piRNAs comple

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organisms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible function of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover.IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesv

A growing number of studies indicate that host species-specific and virus strain-specific interactions of viral molecules with the host innate immune system play a pivotal role in determining virus host range and virulence. Because interacting proteins are likely constrained in their evolution, mutations that are selected to improve virus replication in one species may, by chance, alter the ability of a viral antagonist to inhibit immune responses in hosts the virus has not yet encountered. Based on recent findings of host-species interactions of poxvirus, herpesvirus, and influenza virus proteins, we propose a model for viral fitness and host range which considers the full interactome between a specific host species and a virus, resulting from the combination of all interactions, positive and negative, that influence whether a virus can productively infect a cell and cause disease in different hosts.

STING ER exit is the rate-limiting step in STING signaling, but the mechanism that drives this process is not understood. Paludan and colleagues identify CxORF56, called STEEP here, as a positive regulator of STING signaling.STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Most people living with HIV (PLWH) are coinfected with cytomegalovirus (CMV). Subclinical CMV replication is associated with immune dysfunction and with increased HIV DNA in antiretroviral therapy (ART)-naive and -suppressed PLWH. To identify immunological mechanisms by which CMV could favor HIV persistence, we analyzed 181 peripheral blood mononuclear cell (PBMC) samples from 64 PLWH starting ART during early HIV infection with subsequent virologic suppression up to 58 months. In each sample, we measured levels of CMV and Epstein-Barr virus (EBV) DNA by droplet digital PCR (ddPCR). We also measured expression of immunological markers for activation (HLA-DR+ CD38+), cycling (Ki-67+), degranulation (CD107a+), and the immune checkpoint protein PD-1 on CD4+ and CD8+ T cell memory subsets. Significant differences in percentages of lymphocyte markers by CMV/EBV shedding were identified using generalized linear mixed-effects models. Overall, CMV DNA was detected at 60/181 time points. At the time of ART initiation, the presence of detectable CMV DNA was associated with increased CD4+ T cell activation and CD107a expression and with increased CD8+ T cellular cycling and reduced CD107a expression on CD8+ T cells. While some effects disappeared during ART, greater CD4+ T cell activation and reduced CD107a expression on CD8+ T cells persisted when CMV was present (P < 0.01). In contrast, EBV was not associated with any immunological differences. Among the covariates, peak HIV RNA and CD4/CD8 ratio had the most significant effect on the immune system. In conclusion, our study identified immune differences in PLWH with detectable CMV starting early ART, which may represent an additional hurdle for HIV cure efforts.IMPORTANCE Chronic viral infections such as with HIV and CMV last a lifetime and can continually antagonize the immune system. Both viruses are associated with higher expression of inflammation markers, and recent evidence suggests that CMV may complicate efforts t

Microbes drive most ecosystems and are modulated by viruses that impact their lifespan, gene flow, and metabolic outputs. However, ecosystem-level impacts of viral community diversity remain difficult to assess due to classification issues and few reference genomes. Here, we establish an ∼12-fold expanded global ocean DNA virome dataset of 195,728 viral populations, now including the Arctic Ocean, and validate that these populations form discrete genotypic clusters. Meta-community analyses revealed five ecological zones throughout the global ocean, including two distinct Arctic regions. Across the zones, local and global patterns and drivers in viral community diversity were established for both macrodiversity (inter-population diversity) and microdiversity (intra-population genetic variation). These patterns sometimes, but not always, paralleled those from macro-organisms and revealed temperate and tropical surface waters and the Arctic as biodiversity hotspots and mechanistic hypotheses to explain them. Such further understanding of ocean viruses is critical for broader inclusion in ecosystem models.

Aves polyomavirus 1, psittacine beak and feather disease virus, and psittacid herpesvirus 1 are important pathogens of psittacine birds with the potential to cause substantial morbidity and mortality. Using publically available nucleotide sequences, we developed and validated a triplex real-time PCR (rtPCR) assay to rapidly detect these 3 viruses. The assay had high analytical sensitivity, detecting <6 copies of viral DNA per reaction, and 100% analytical specificity, showing no cross-reactivity with 59 other animal pathogens. Archived formalin-fixed, paraffin-embedded tissues from psittacine birds diagnosed at postmortem as infected with each of the viruses as well as virus-negative birds were used to validate the utility of the assay. Birds were selected for the positive cohort if they showed histologic evidence of infection (i.e., characteristic inclusion bodies in tissues); birds in the negative cohort had final diagnoses unrelated to the pathogens of interest. The triplex rtPCR assay confirmed 98% of histopathology-positive cases, and also identified subclinical infections that were not observed by histologic examination, including coinfections. Birds that tested positive only by rtPCR had significantly higher cycle threshold values compared to those with histologic evidence of infection. Positive, negative, and overall percentage agreements as well as the kappa statistic between the results of the assay and histopathology were high, demonstrating the usefulness of the assay as a tool to confirm disease diagnoses, and to improve detection of subclinical infections.

Our genomes are dominated by repetitive elements. The majority of these elements derive from retrotransposons, which expand throughout the genome through a process of reverse transcription and integration. Short interspersed nuclear elements, or SINEs, are an abundant class of retrotransposons that are transcribed by RNA polymerase III, thus generating exclusively noncoding RNA (ncRNA) that must hijack the machinery required for their transposition. SINE loci are generally transcriptionally repressed in somatic cells but can be robustly induced upon infection with multiple DNA viruses. Recent research has focused on the gene expression and signaling events that are modulated by SINE ncRNAs, particularly during gammaherpesvirus infection. Here, we review the biology of these SINE ncRNAs, explore how DNA virus infection may lead to their induction, and describe how novel gene regulatory and immune-related functions of these ncRNAs may impact the viral life cycle.

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size.

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Viruses are the most abundant members of the human oral microbiome, yet relatively little is known about their biodiversity in humans. To improve our understanding of the DNA viruses that inhabit the human oral cavity, we examined saliva from a cohort of eight unrelated subjects over a 60-day period. Each subject was examined at 11 time points to characterize longitudinal differences in human oral viruses. Our primary goals were to determine whether oral viruses were specific to individuals and whether viral genotypes persisted over time. We found a subset of homologous viral genotypes across all subjects and time points studied, suggesting that certain genotypes may be ubiquitous among healthy human subjects. We also found significant associations between viral genotypes and individual subjects, indicating that viruses are a highly personalized feature of the healthy human oral microbiome. Many of these oral viruses were not transient members of the oral ecosystem, as demonstrated by the persistence of certain viruses throughout the entire 60-day study period. As has previously been demonstrated for bacteria and fungi, membership in the oral viral community was significantly associated with the sex of each subject. Similar characteristics of personalized, sex-specific microflora could not be identified for oral bacterial communities based on 16S rRNA. Our findings that many viruses are stable and individual-specific members of the oral ecosystem suggest that viruses have an important role in the human oral ecosystem.

Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cell's chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles.

Double-stranded (ds) DNA viruses are often described as evolving through long-term codivergent associations with their hosts, a pattern that is expected to be associated with low rates of nucleotide substitution. However, the hypothesis of codivergence between dsDNA viruses and their hosts has rarely been rigorously tested, even though the vast majority of nucleotide substitution rate estimates for dsDNA viruses are based upon this assumption. It is therefore important to estimate the evolutionary rates of dsDNA viruses independent of the assumption of host-virus codivergence. Here, we explore the use of temporally structured sequence data within a Bayesian framework to estimate the evolutionary rates for seven human dsDNA viruses, including variola virus (VARV) (the causative agent of smallpox) and herpes simplex virus-1. Our analyses reveal that although the VARV genome is likely to evolve at a rate of approximately 1 x 10(-5) substitutions/site/year and hence approaching that of many RNA viruses, the evolutionary rates of many other dsDNA viruses remain problematic to estimate. Synthetic data sets were constructed to inform our interpretation of the substitution rates estimated for these dsDNA viruses and the analysis of these demonstrated that given a sequence data set of appropriate length and sampling depth, it is possible to use time-structured analyses to estimate the substitution rates of many dsDNA viruses independently from the assumption of host-virus codivergence. Finally, the discovery that some dsDNA viruses may evolve at rates approaching those of RNA viruses has important implications for our understanding of the long-term evolutionary history and emergence potential of this major group of viruses.

The capsids of large, icosahedral dsDNA viruses are built from well-ordered aggregates of capsomers, known as trisymmetrons and pentasymmetrons, which are centered on the icosahedral 3-fold and 5-fold axes, respectively. We derive the complete set of rules for constructing an icosahedral structure from these symmetrons when the T lattice symmetry is odd and show that there are three classes of solutions, each of which follows from a different relationship between the size of the pentasymmetron and the values of the h and k icosahedral lattice parameters. Together, these three classes account for all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons. Also, every icosahedral lattice with odd T number has solutions from exactly two of these three classes, with the set of allowed classes dependent on which of the two lattice parameters is odd. For symmetric lattices (if h=k or h=0), the two solutions yield the same symmetron sizes, but when the lattice parameters are equal (h=k) the solutions can be distinguished by the relative orientations of the symmetrons. We discuss these results in the context of known virus structures and explore the implications for viruses whose structures have not yet been solved.

Wild-type viruses with intrinsic oncolytic capacity in human includes DNA viruses like some autonomous parvoviruses and many RNA viruses. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance. It includes limited virus spread in tumor masses, stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limited tumor transduction, immune-mediated inactivation or destruction of the virus. For replication-competent vectors without approved antiviral agents, suicide genes might be used as fail-safe mechanism. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Therefore, viruses that target the defective self-renewal pathways in cancer cells might lead to improved outcomes.In this thesis, data we generated in the field of oncolytic autonomous parvoviruses are presented.We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population, (CAT ELISA) or at the single cell level, (FACS analysis of Green Fluorescent Protein). Cat expression was substantial (up to 10000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells, (with the exception of an expression slightly above background in fibroblasts.). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP positive normal cells could be detected with the exception of very few (less than 0, Doctorat en sciences, Spécialisation biologie moléculaire, info:eu-repo/semantics/nonPublished

This thesis explores the population genetics of the baculovirus infection process and the consequences for virus evolution. Using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and lepidopteran insect larvae as a model system, we attempt to characterize (1) elemental virus-host and virus-virus interactions pertinent to virus invasion and disease of the host, focussing largely on the number of virus individuals invading a host insect and including the first strong experimental evidence that a single virion can cause disease in a host animal, and (2) the interactions that can occur between virus genotypes during the process of diseasing the host, specifically competition. The development of two technologies was necessary to address these questions. First, a method for generating clonal baculovirus populations containing molecular tag sequences was devised: insect larvae were transfected with full-length baculovirus genomic DNA modified to allow replication in Escherichia coli (bacmids). Second, a quantitative real-time PCR (qPCR) based assay was developed to measure the frequency of a baculovirus genotype in a mixed population. In order to estimate the number of pathogen individuals invading the host and causing death, a basic probabilistic model that links this number to host survival was developed and experimentally tested. This model is based on the independent action hypothesis (IAH): the notion that (1) every pathogen individual has a fixed probability of invading the host, (2) if a pathogen individual invades the host this will irrevocably lead to host death, and (3) that pathogen individuals act independently. Here the model was specifically developed to predict how often hosts are invaded by two pathogen genotypes, when being challenged by a pathogen population consisting of two genotypes. Model predictions were tested using two near-identical bacmid-derived virus genotypes, larvae of three host species, and the qPCR assay. IAH model predicti

English: Incidences of an Adenoviral infection have been recently reported in South African psittacine birds. These birds have been diagnosed by histopathology post-mortem. This Psittacine Adenovirus (PsAdV) has been reported as the second most deadly disease-causing virus in psittacine species with Psittacine Beak and Feather Disease Virus (PBFD) being the first. High mortalities with no symptoms have been observed amongst the birds. There is limited information on the distribution and antigenic characterisation of PsAdV in South Africa. This prompted the investigation into the isolation and characterisation of this PsAdV by virtue of molecular and conventional techniques. Two birds suspected of being exposed to PsAdV were donated to the laboratory. Histopathological tests were already performed on these birds by a consulting veterinarian who found evidence supporting the Adenoviral infection. The livers of the parrots received were homogenised and DNA was then extracted from the suspension. These birds were from the same parrot breeding farm in the Free State region and their deaths occurred around September a year apart, inferring a seasonal occurrence of the infected birds. A polymerase chain reaction (PCR) was established as a rapid test to confirm the Adenoviral infection amongst the birds received. A primer pair amplifying the hexon gene loop1 variable region (L1) located in two conserved pedestral regions was modified using an existing primer pair as this gene codes for the hexon protein where the group, subgroup and type specific antigenic determinants are located. PCR resulted in the expected amplicon size of ~587bp for all the samples tested with the use of a Fowl Adenovirus (FAdV) received from the Faculty of Veterinary Science, Onderstepoort (Pretoria) as a positive control. The positive DNA products were then subjected to restriction fragment length polymorphism (RFLP) in order to investigate differences in the restriction profiles. The suspected PsAdV, Afrikaans: Gevalle van Adenovirale infeksies is onlangs in Suid-Afrikaanse psittacine voëls aangemeld. Diagnose van die voëls was gedoen deur na-doodse histologiese ondersoeke. Die Psittacine Adenovirus (PsAdV) word beskou as die tweede mees dodlikste virus in psittacine spesies met Psittacine Beak and Feather Disease virus (PBFD) die dodlikste. Daar is tans beperkte inligting beskikbaar ten opsigte van die verspreiding en die antigeniese karakterisering van die PsAdV in Suid-Afrika. Dit het gelei tot die isolasie en die karakterisering van die PsAdV deur molekulêre en konvensionele tegnieke. Twee voëls wat moontlik blootgestel was aan PsAdV is geskenk aan dié laboratorium. Histopatalogiese toetse was reeds op die voëls uitgevoer deur ‘n veearts wat Adenovirale infeksie simptome aangemeld het. Die voëls was afkomstig vanaf dieselfde papegaai broeiplaas in die Vrystaat omgewing en beide is dood in September ‚ ’n jaar uitmekaar, wat moontlik ‘n seisoenale infeksie aandui. DNA is geëkstraheer uit gehomogeniseerde lewers van die papegaaie en ’n polimerasee ketting reaksie (PKR) gebruik as metode vir 'n vinnige diagnose van Adenovirale infeksie. ‘n Preimstuk paar wat die hexon geen boog 1 veranderlike gebied (L1), teenwoordig in twee gekonserveerde areas, is ontwerp aangesien die geen vir die hexon proteïen kodeer. Die groep, subgroep en tipe spesifieke antigeen bepalers word deur hierdie protein bepaal. ’n Verwagte produk grootte van ongeveer 587 bp is verkry vir al die monsters getoets. ’n Hoender Adenovirus (FAdV), ontvang van die Fakulteit van Veeartsenykundige wetenskappe, Onderstepoort (Pretoria) is gebruik as positiewe kontrole. Restriksie Fragment Lengte Polimorfisme (RFLP) is daaropvolgend gebruik om die verskille in die restriksie profiele van die positiewe DNA produkte te ondersoek. Die verdagte PsAdV monsters het dieselfde profiele gehad en die profiel was verskillend van die profiel verkry vanaf die hoender monster. Die PKR produk wat verkry is van die geïnf, National Research Foundation (NRF)

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146¿bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

The sequences of two giant viral genomes, Mimivirus and a polydnavirus, have recently been published. Mimivirus has the largest known viral genome and encodes an unprecedented number of proteins, whereas the polydnavirus genome has an extremely low coding density and does not encode DNA-replication proteins. These and other unusual features challenge the way we view the evolution and definition of viruses.

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

This work presents ultrastructural observations of virus-like particles in the sporogonous cycle of P. berghei, P. c. bastianelli and P. gallinaceum as well as in ookinete preparations of P. berghei. The particles are about 50 nm in diameter, are sometimes arranged in a geometric crystal configuration and have a limiting membrane or envelope. A curious staining phenomenon suggests that the viral nucleoid is DNA containing. The possiblility that the virus-like particles may be involved in early vacuolation of the differentiating oocyst is discussed. Further attempts to characterize the virus-like particles were inconclusive. As an interesting by-product of these investigations, observations of P. berghei ookinete and gametocyte forms were made.

The mechanisms of inactivation of aerosolized bacteriophage phiX174 in atmospheres containing ozone, cyclohexene, or ozonized cyclohexene were studied by using 32P-labelled phage. The inactivation of the aerosolized phage in clean air or in air containing cyclohexene is due to damage of the protein coat since the deoxyribonucleic acid (DNA) extracted from the inactivated phage retains its biological activity. Inactivation of the phage in air containing ozone is mainly due to protein damage whereas inactivation in air containing ozonized cyclohexene is due both to protein and DNA damage. Sucrose gradient analysis shows that aerosolized inactivated phiX 174 releases unbroken DNA. In contrast, the DNA from phage phiX 174 inactivated by ozonized cyclohexene is broken. The inactivation of aerosolized phage phiX 174-DNA was studied in the same atmospheres using 32P-labelled DNA. phiX 174-DNA aerosolized in clean air or air containing cyclohexene at 75% r.h. is inactivated by a factor of 2 in 30 min. The inactivated DNA is broken. Ozone as well as ozonized cyclohexene inactivates DNA very fast causing breaks in the molecule. This is in contrast with the intact bacteriophage in which ozone does not produce breaks in the DNA. Chemicals/CAS: ozone, 10028-15-6; Aerosols; Alkenes; DNA, Viral; Ozone, 10028-15-6

The first demonstrations that cancer could have an infectious nature was by Ellerman and Bang (1) ~ who showed that leukemia in chickens was transmissible with cell-free extracts and by Rous (2), who found in a similar fashion that naturally occurring chicken sarcomas were transmissible. Although they were able to show that these cell-free extracts contained a transmissible agent~ the idea that this induced cancer was received by the scientific world at that time with great skepticism. The interest in oncogenic viruses was strongly enhanced in the early 60's by the isolation of mammalian tumor viruses and the general acceptance that at least some of these viruses were tumorigenic. The discovery of the reverse transcriptase enzyme in RNA tumor viruses (3,4), gave a logical explanation for how these viruses became integrated in the chromosomes of eukaryotic cells. Taxonomically, oncogenic viruses are members of diverse families. DNA viruses (herpes-, adeno- and papovaviruses) as well as many members of the retrovirus family (containing RNA such as the type C RNA viruses) are capable of inducing tumors. For the retroviruses two different routes to become transforming (oncogenic) have become clear. The majority of these viruses (the acute type C RNA transforming viruses) 11 acquire11 certain genetic sequences (oncogenes) from their host, which are necessary to initiate and maintain the malignant transformation of the cell by the virus. Other retroviruses integrate their genome nearby oncogenic sequences in the chromosome of their host. Independent of the exact mechanism, these viruses share the capability of inducing tumorigenesis by triggering the transcription of certain sequences, and it is the proteins encoded by these sequences which are necessary to maintain the neoplastic phenotype of the infected cell The accumulating number of independent isolates of tumorigenic retroviruses induced in the mid-70's a worldwide search for these viruses in humans. Only very recen

The restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and pyrimidine isostich analysis. The Hind and HaeIII fragments were ordered relative to each other by cross digestion and a physical map of the S13 genome constructed. Comparison of the Hind cleavage patterns of S13 RF DNA and X174 RF DNA showed the majority of the fragments from the two DNAs coincided with each other except for three of the thirteen S13 fragments and three of the thirteen X174 fragments. Comparison of the HaeIII patterns of the two DNAs revealed a lack of coincidence of one S13 fragment only and two X174 fragments. From the data obtained by the cleavage of the two DNAs by Hind and HaeIII, a correlation between the physical restriction enzyme cleavage maps and the genetic map of the two phages was made. The differences in cleavage of the two DNAs by the restriction enzymes have been explained by changes in two restriction enzyme sites in the AB region and one change of site in the F region of the genetic map of the two bacteriophages.

Thèse d'agrégation de l'enseignement supérieur (Faculté de médecine) -- UCL, 1963