The Bacterial Symbiont Wolbachia Induces Resistance to RNA Viral Infections in Drosophila melanogaster

Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia–host interaction.     

Complement C4 Prevents Viral Infection through Capsid Inactivation

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Infection avoidance behavior: Viral exposure reduces the motivation to forage in female Drosophila melanogaster

Infection avoidance behaviors are the first line of defense against pathogenic encounters. Behavioral plasticity in response to internal or external cues of infection can therefore generate potentially significant heterogeneity in infection. We tested whether Drosophila melanogaster exhibits infection avoidance behavior, and whether this behavior is modified by prior exposure to Drosophila C Virus (DCV) and by the risk of DCV encounter. We examined 2 measures of infection avoidance: (1) the motivation to seek out food sources in the presence of an infection risk and (2) the preference to land on a clean food source over a potentially infectious source. While we found no evidence for preference of clean food sources over potentially infectious ones, previously exposed female flies showed lower motivation to pick a food source when presented with a risk of encountering DCV. We discuss the relevance of behavioral plasticity during foraging for host fitness and pathogen spread.     

Adaptation to the laboratory environment in Drosophila subobscura

Adaptation to a novel environment is expected to have a number of features. Among these is a temporal increase in fitness and some or all of its components. It is also expected that additive genetic variances for these fitness characters will fall. Finally, it is expected that at least some additive genetic correlations will decrease, from positive toward negative values. In a study of several life‐history variables in a Drosophila subobscura population sampled from the wild and then cultured in the laboratory, we did not find any such longitudinal trends over the first 29 generations. However, a temporal comparison (over 14 generations) of the later generations of this laboratory‐adapted population with a new population, derived from a more recent wild‐caught sample, indicated clearly that laboratory adaptation was nonetheless occurring. This study suggests the need for extensive replication and control in studies of the features of adaptation to a novel environment.     

Faster Adaptation in Smaller Populations: Counterintuitive Evolution of HIV during Childhood Infection

Analysis of HIV-1 gene sequences sampled longitudinally from infected individuals can reveal the evolutionary dynamics that underlie associations between disease outcome and viral genetic diversity and divergence. Here we extend a statistical framework to estimate rates of viral molecular adaptation by considering sampling error when computing nucleotide site-frequencies. This is particularly beneficial when analyzing viral sequences from within-host viral infections if the number of sequences per time point is limited. To demonstrate the utility of this approach, we apply our method to a cohort of 24 patients infected with HIV-1 at birth. Our approach finds that viral adaptation arising from recurrent positive natural selection is associated with the rate of HIV-1 disease progression, in contrast to previous analyses of these data that found no significant association. Most surprisingly, we discover a strong negative correlation between viral population size and the rate of viral adaptation, the opposite of that predicted by standard molecular evolutionary theory. We argue that this observation is most likely due to the existence of a confounding third variable, namely variation in selective pressure among hosts. A conceptual non-linear model of virus adaptation that incorporates the two opposing effects of host immunity on the virus population can explain this counterintuitive result.     

Exercise Improves Host Response to Influenza Viral Infection in Obese and Non-Obese Mice through Different Mechanisms

Obesity has been associated with greater severity of influenza virus infection and impaired host defense. Exercise may confer health benefits even when weight loss is not achieved, but it has not been determined if regular exercise improves immune defense against influenza A virus (IAV) in the obese condition. In this study, diet-induced obese mice and lean control mice exercised for eight weeks followed by influenza viral infection. Exercise reduced disease severity in both obese and non-obese mice, but the mechanisms differed. Exercise reversed the obesity-associated delay in bronchoalveolar-lavage (BAL) cell infiltration, restored BAL cytokine and chemokine production, and increased ciliary beat frequency and IFNα-related gene expression. In non-obese mice, exercise treatment reduced lung viral load, increased Type-I-IFN-related gene expression early during infection, but reduced BAL inflammatory cytokines and chemokines. In both obese and non-obese mice, exercise increased serum anti-influenza virus specific IgG2c antibody, increased CD8+ T cell percentage in BAL, and reduced TNFα by influenza viral NP-peptide-responding CD8+ T cells. Overall, the results suggest that exercise “restores” the immune response of obese mice to a phenotype similar to non-obese mice by improving the delay in immune activation. In contrast, in non-obese mice exercise treatment results in an early reduction in lung viral load and limited inflammatory response.     

Adaptation of Drosophila to temperature: Heat-shock proteins and survival in Drosophila melanogaster

Two stocks of Drosophila melanogaster, one sensitive (6.5% survival) and one resistant (76.24%) to heat shock (40°C/25 min) were derived through indirect selection [1]. Genetic analysis of heat-sensitive and heat-resistant lines we had selected revealed that the survival rate is chiefly determined by cytoplasmic inheritance but also depends to some extent on the nucleus [1]. The ability of the fly to survive thermal stress was found to have an excellent correlation with the kinetics of protein synthesis in ovaries or glands subjected to heat treatment. The incorporation rate of ³⁵S-methionine into proteins was found to be higher for strains exhibiting higher survival (R₁, R₁S₁) than for strains with a lesser ability (S₁, S₁ R₁) to survive heat shock. Moreover, the intensity of labeling of the proteins synthesized and especially of the hsps (heat-shock proteins) after the heat shock is higher in the R₁ and R₁S₁ stocks than in the S₁ and S₁R₁ stocks. This convergence between survival and the cellular level of hsps (both manipulated by selection) bears on the physiological significance of these proteins which seems to participate in the control of the survival as an additive component.     

Adaptation to P element transposon invasion in Drosophila melanogaster

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.     

Envelope Vaccination Shapes Viral Envelope Evolution following Simian Immunodeficiency Virus Infection in Rhesus Monkeys

The evolution of envelope mutations by replicating primate immunodeficiency viruses allows these viruses to escape from the immune pressure mediated by neutralizing antibodies. Vaccine-induced anti-envelope antibody responses may accelerate and/or alter the specificity of the antibodies, thus shaping the evolution of envelope mutations in the replicating virus. To explore this possibility, we studied the neutralizing antibody response and the envelope sequences in rhesus monkeys vaccinated with either gag-pol-nef immunogens or gag-pol-nef immunogens in combination with env and then infected with simian immunodeficiency virus (SIV). Using a pseudovirion neutralization assay, we demonstrate that envelope vaccination primed for an accelerated neutralizing antibody response following virus challenge. To monitor viral envelope evolution in these two cohorts of monkeys, full-length envelopes from plasma virus isolated at weeks 37 and 62 postchallenge were sequenced by single genome amplification to identify sites of envelope mutations. We show that env vaccination was associated with a change in the pattern of envelope mutations. Prevalent mutations in sequences from gag-pol-nef vaccinees included deletions in both variable regions 1 and 4 (V1 and V4), whereas deletions in the env vaccinees occurred only in V1. These data show that env vaccination altered the focus of the antibody-mediated selection pressure on the evolution of envelope following SIV challenge.Immune containment of human immunodeficiency virus (HIV-1) is complicated by the continuous genetic evolution of the virus. The evolution of the HIV-1 envelope is shaped, in part, by selective pressure of neutralizing antibodies (6, 12, 27, 34-36, 40). Changes in envelope sequence and glycosylation patterns following infection can allow the virus to escape neutralization. If the rate and extent of envelope sequence evolution following infection can be decreased, immune containment of HIV-1 may be improved.One possible strategy for modifying envelope evolution is vaccination prior to infection. A vaccine-elicited memory immune response could focus and potentiate the humoral immune response that develops following infection. The possible consequence of vaccination has not been assessed, however, because of the limited number of human volunteers who have received highly immunogenic envelope immunogens and subsequently became infected with HIV-1.Simian immunodeficiency virus (SIV) infection of rhesus monkeys provides a powerful model to study the effect of vaccination on envelope evolution. Like HIV-1, SIV employs both the CD4 molecule and the chemokine receptor CCR5 to enter a target cell and cause an AIDS-like disease in macaques (16, 22). Both SIV and HIV-1 envelopes are heavily glycosylated, with approximately 50% of their mass derived from carbohydrates (14, 21). SIV and HIV-1 envelopes share approximately 40% amino acid homology (10, 11) and have overlapping variable and constant regions, although the variable region 3 (V3) of HIV-1 envelope does not align with the homologous region of SIV envelope (7). Following SIV infection in rhesus monkeys, SIV envelope evolves most rapidly in variable regions 1 and 4 (V1 and V4, respectively), leading to nucleotide additions, deletions, and/or mutations that can potentially translate to changes in glycosylation (7, 9, 13, 15, 19, 29, 30).Studies done to characterize SIV neutralization suggest that it occurs through mechanisms similar to those seen in HIV-1 neutralization. Amino acid mutations in the envelope of both viruses contribute to the evasion of antibody binding directly by changing recognition sequences and/or envelope conformation. In addition, the glycosylation of envelope serves as a further obstacle to antibody recognition (20, 33, 40). Considerable effort has been devoted to defining neutralizing epitopes of the HIV and SIV envelopes. The known neutralizing human monoclonal antibodies elicited during natural infection are directed against HIV-1 envelope target sites on both gp120 and gp41, including the V3 region, the CD4 binding site, oligomannose residues of gp120, and gp41 (17, 31). The neutralizing epitope profile of SIV envelope includes the CD4 binding site and gp41 but not the V3 region. There is conflicting evidence as to whether V1, V2, and/or V4 of SIV are targets for antibody neutralization (15, 18, 19). The present study addresses whether vaccine-induced immune responses accelerate the generation of autologous neutralizing antibodies following SIV challenge in rhesus monkeys and how this humoral immune response can potentially shape viral sequence evolution.     

Enhanced CD8 T Cell Responses through GITR-Mediated Costimulation Resolve Chronic Viral Infection

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.     

Experimental Evolution of a Plant Pathogen into a Legume Symbiont

Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.     

Viral Infection and Cell Differentiation

The dependence of viral reproduction and success of viral infection on cell differentiation is briefly reviewed at the example of picornaviruses—small RNA viruses of animals. In particular, the role of the cell factors in viral tropism, control of viral RNA translation, and the pattern of infected cell death are discussed.