Molecular Detection, Penetrance, and Transmission of an Inherited Virus Responsible for Behavioral Manipulation of an Insect Parasitoid

For insects, the prevalence of numerous vertically transmitted viruses can be high in their host populations. These viruses often have few, if any, pathological effects on their hosts, and consequently, many of them can remain unnoticed for long periods, despite their potential role in the evolution of the host phenotype. Some females of Leptopilina boulardi, a solitary parasitoid of Drosophila larvae, are infected by an inherited virus (LbFV) that manipulates the behavior of the wasp by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). This behavioral alteration allows horizontal transmission of the virus within superparasitized Drosophila larvae. Using suppressive subtractive hybridization with infected and uninfected lines, we identified one putative viral sequence. Based on this sequence, we developed a simple PCR test. We tested the correlation between the superparasitism phenotype and PCR amplification of the putative viral marker using several experimental conditions (including horizontal transfers) and several parasitoid genotypes. All of the results revealed that there was a perfect match between the superparasitism phenotype and the amplification profile, which validated use of the molecular marker as a tool to track the presence of the virus and provided the first genomic data for this fascinating virus. The results also show that there was very efficient horizontal and vertical transmission of LbFV, which probably explains its high prevalence in the French populations that we sampled (67 and 70% of infected females). This manipulative virus is likely to play a major role in the ecology and evolution of its parasitoid host.     

Phage spread dynamics in clonal bacterial populations is depending on features of the founder cell

Plate-cultured bacterial colonies are intriguing models to study host-parasite interactions in senescent populations. During the growth of bacteriophage-infected colonies there is a synchronous prophage induction episode among lysogenic cells that allows a dramatic but time-restricted amplification of viral particles. We report here that the dynamics of phage spread depends on the history of the lysogenic cell that establishes the clonal population, the duration of the pre-burst period being shorter when the founder, infected cell derives from older colonies. These results offer a physiologic explanation for the self-contained progression of the viral spread in closed environments, that ensures both viral dissemination but also survival of most of the host cells.     

Viral control of bacterial biodiversity – evidence from a nutrient-enriched marine mesocosm experiment

We demonstrate here results showing that bottom-up and top-down control mechanisms can operate simultaneously and in concert in marine microbial food webs, controlling prokaryote diversity by a combination of viral lysis and substrate limitation. Models in microbial ecology predict that a shift in the type of bacterial growth rate limitation is expected to have a major effect on species composition within the community of bacterial hosts, with a subsequent shift in the composition of the viral community. Only moderate effects would, however, be expected in the absolute number of coexisting virus–host pairs. We investigated these relationships in nutrient-manipulated systems, under simulated in situ conditions. There was a strong correlation in the clustering of the viral and bacterial community data supporting the existence of an important link between the bacterial and viral communities. As predicted, the total number of viral populations was the same in all treatments, while the composition of the viral community varied. Our results support the theoretical prediction that there is one control mechanism for the number of niches for coexisting virus–host pairs (top-down control), and another mechanism that controls which virus–host pairs occupy these niches (bottom-up control).     

INHERITANCE AND MEIOTIC ELIMINATION OF A VIRUS GENOME IN THE HOST ECTOCARPUS SZLZCULOSUS (PHAEOPHYCEAE)1

The marine brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye is frequently infected by a latent DNA virus that multiplies in modified sporangia and gametangia of the host. We describe a polymerase chain reaction (PCR) procedure for the amplification and detection of viral DNA in Ectocarpus. PCR analysis of parents and progeny plants confirmed that virus DNA passes through meiosis like a Mendelian trait. An infected sporophyte produced equal numbers of gametophytes with and without the viral genome. Thus, meiosis in sexual populations of the host acts as a mechanism for the creation of virus-free progeny.     

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ~1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.     

Drosophila Adaptation to Viral Infection through Defensive Symbiont Evolution

Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. Here we show that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit.     

Efficient method to isolate and purify viruses of bacteria from marine environments

Aim:  To isolate viruses of specific heterotrophic bacterial strains from marine environments using a host addition/virus amplification protocol (HAVAP) for use in phage/host systems.
Methods and Results:  Bacteria-free seawater samples containing natural viruses assemblages were inoculated with a single laboratory grown bacterial host of interest in a nutrient-enriched [peptone, Fe(III) and yeast extract] seawater suspension. These conditions enhanced the replication of only those virus(s) capable of infecting the host bacterium. After incubation, free viruses were recovered at concentrations ranging 10⁵–10¹⁰ infectious virus particles per ml of seawater. Using this approach, 15 viruses were isolated and represented 12 unique phage/host systems. Two of the hosts tested were infected by more than one virus.
Conclusions:  Isolation of high concentrations of specific viruses is possible even if their initial concentrations in native waters are low. This approach allows the recovery of phage/host systems that may not be numerically dominant.
Significance and Impact of the Study:  This host enrichment protocol for virus detection and isolation is well-suited for aquatic viral ecology studies that require phage/host systems.     

Do virus‐resistant plants pose a threat to non‐target ecosystems? II. Risk assessment of an Australian pathosystem using multi‐scale field experiments

It has been widely argued that the acquisition of novel disease resistance genes by wild host populations following the release of novel pathogen‐resistant plants into agricultural systems could pose a significant threat to non‐target plant communities. However, predicting the magnitude of ecological release in wild plant populations following the removal of disease remains a major challenge. In this paper we report on the second phase of a tiered risk assessment designed to investigate the role of disease on host growth, survival, fecundity and fitness in a model pathosystem (the pasture species Trifolium repens infected with Clover yellow vein virus, ClYVV) and to assess the level of risk posed to at‐risk native plant communities in southeast Australia by newly developed genetically modified and conventionally bred virus‐resistant T. repens genotypes. Multi‐year field experiments conducted in woodland and grassland environments using host‐pathogen arrays derived from 14 ClYVV isolates and 21 T. repens genotypes indicate that viral infection reduces fecundity, growth and survival of wild T. repens plants but that the severity of these effects depends on host tolerance to infection, isolate aggressiveness and specific spatial and temporal environmental conditions. Demographic modelling showed that by reducing host survival and growth, ClYVV also limits the intrinsic population growth rate and niche size of wild T. repens populations. Given the significant fitness cost associated with viral infection we conclude that virus‐resistant T. repens genotypes may pose a threat to some high conservation‐value non‐target ecosystems in SE Australia. We also argue that long‐term, multi‐tiered experiments conducted in a range of controlled and non‐controlled environments are necessary to detect and accurately quantify risks associated with the release of disease‐resistant plants in general.     

The recent spread of a vertically transmitted virus through populations of Drosophila melanogaster

The sigma virus is a vertically transmitted pathogen that commonly infects natural populations of Drosophila melanogaster. This virus is the only known host-specific pathogen of D. melanogaster, and so offers a unique opportunity to study the genetics of Drosophila-viral interactions in a natural system. To elucidate the population genetic processes that operate in sigma virus populations, we collected D. melanogaster from 10 populations across three continents. We found that the sigma virus had a prevalence of 0-15% in these populations. Compared to other RNA viruses, we found that levels of viral genetic diversity are very low across Europe and North America. Based on laboratory measurements of the viral substitution rate, we estimate that most European and North American viral isolates shared a common ancestor approximately 200 years ago. We suggest two explanations for this: the first is that D. melanogaster has recently acquired the sigma virus; the second is that a single viral type has recently swept through D. melanogaster populations. Furthermore, in contrast to Drosophila populations, we find that the sigma viral populations are highly structured. This is surprising for a vertically transmitted pathogen that has a similar migration rate to its host. We suggest that the low structure in the viral populations can be explained by the smaller effective population size of the virus.     

Virulence variability of the Drosophila C virus and effects of the microparasite on demographic parameters of the host (Drosophila melanogaster)

We carried out experiments with the Drosophila C virus (DCV), a nonhereditary virus acting on demographic parameters of infected Drosophila host populations. It is well known that DCV increases mortality rate, decreases developmental time, and increases daily fecundity. As usual for Drosophila viruses, the DCV was multiplied in vivo. In this study we tested the hypothesis of virulence variability in DCV strains by isolating different stocks of the virus. The flies were tested for susceptibility to injection of such isolates and for virulence variability. Possible interactions between demographic parameters in three Drosophila host populations and injected isolates were studied under two egg densities (low and high). The hypothesis of virulence variability of DCV was supported by significant differences in mortality rates, depending on whether virus isolates were ingested or injected. When DCV was ingested, differences between host mortality rates were independent of the Drosophila host populations. Nevertheless, the developmental time was equally decreased by each virus isolate, independent of the host population. Moreover, the two viral stocks strongly increased the egg production of the flies. This experimental approach clearly showed that DCV could be considered a polymorphic virus. The phenotypic interactions between DCV and host flies varied according to parasite genotype.     

Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B′) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B′ genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B′, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as “viritopes” to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.     

Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.     

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.     

High-throughput analysis of growth differences among phage strains

Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments.     

Replication of viruses in a growing plaque: a reaction-diffusion model.

An understanding of the viral replication process commonly referred to as "plaque growth" is developed in the context of a reaction-diffusion model. The interactions among three components: the virus, the healthy host, and the infected host are represented using rates of viral adsorption and desorption to the cell surface, replication and release by host lysis, and diffusion. The solution to the full model reveals a maximum in the dependence of the velocity of viral propagation on its equilibrium adsorption constant, suggesting that conditions can be chosen where viruses which adsorb poorly to their hosts will replicate faster in plaques than those which adsorb well. Analytic expressions for the propagation velocity as a function of the kinetic and diffusion parameters are presented for the limiting cases of equilibrated adsorption, slow adsorption, fast adsorption, and large virus yields. Hindered diffusion at high host concentrations must be included for quantitative agreement with experimental data.     

Stochastic exits from dormancy give rise to heavy‐tailed distributions of descendants in bacterial populations

Variance in reproductive success is a major determinant of the degree of genetic drift in a population. While many plants and animals exhibit high variance in their number of progeny, far less is known about these distributions for microorganisms. Here, we used a strain barcoding approach to quantify variability in offspring number among replicate bacterial populations and developed a Bayesian method to infer the distribution of descendants from this variability. We applied our approach to measure the offspring distributions for five strains of bacteria from the genus Streptomyces after germination and growth in a homogenous laboratory environment. The distributions of descendants were heavy‐tailed, with a few cells effectively ‘winning the jackpot’ to become a disproportionately large fraction of the population. This extreme variability in reproductive success largely traced back to initial populations of spores stochastically exiting dormancy, which provided early‐germinating spores with an exponential advantage. In simulations with multiple dormancy cycles, heavy‐tailed distributions of descendants decreased the effective population size by many orders of magnitude and led to allele dynamics differing substantially from classical population genetics models with matching effective population size. Collectively, these results demonstrate that extreme variability in reproductive success can occur even in growth conditions that are far more homogeneous than the natural environment. Thus, extreme variability in reproductive success might be an important factor shaping microbial population dynamics with implications for predicting the fate of beneficial mutations, interpreting sequence variability within populations and explaining variability in infection outcomes across patients.     

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.     

The role of antigenic stimulation and cytotoxic T cell activity in regulating the long-term immunopathogenesis of HIV: mechanisms and clinical implications

This paper develops a predictive mathematical model of cell infection, host immune response and viral replication that reproduces observed long-term trends in human immunodeficiency virus (HIV) pathogenesis. Cell activation induced by repeated exposure to many different antigens is proposed as the principal mechanism of providing target cells for HIV infection and, hence, of CD4+ T cell depletion, with regulation of the overall T cell pool size causing concomitant CD8 pool increases. The model correctly predicts the cross-patient variability in disease progression, the rate of which is found to depend on the efficacy of anti-HIV cytotoxic T lymphocyte responses, overall viral pathogenicity and random effects. The model also predicts a variety of responses to anti-viral therapy, including episodic residual viral replication and discordant responses and we find that such effects can be suppressed by increasing the potency of treatment.     

Concentration and purification of rubella virus using monolithic chromatographic support

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.