Antiviral protection and the importance of Wolbachia density and: tissue tropism in Drosophila simulans

Wolbachia, a maternally transmitted endosymbiont of insects, is increasingly being seen as an effective biological control agent that can interfere with transmission of pathogens, including dengue virus. However, the mechanism of antiviral protection is not well understood. The density and distribution of Wolbachia in host tissues have been implicated as contributing factors by previous studies with both mosquitoes and flies. Drosophila flies infected with five diverse strains of Wolbachia were screened for the ability to mediate antiviral protection. The three protective Wolbachia strains were more closely related and occurred at a higher density within whole flies than the two nonprotective Wolbachia strains. In this study, to further investigate the relationship between whole-fly Wolbachia density and the ability to mediate antiviral protection, tetracycline was used to decrease the abundance of the high-density, protective Wolbachia strain wAu prior to viral challenge. Antiviral protection was lost when the density of the protective Wolbachia strain was decreased to an abundance similar to that of nonprotective Wolbachia strains. We determined the Wolbachia density and distribution in tissues of the same five fly-Wolbachia combinations as used previously. The Wolbachia density within the head, gut, and Malpighian tubules correlated with the ability to mediate antiviral protection. These findings may facilitate the development of Wolbachia biological control strategies and help to predict host-Wolbachia pairings that may interfere with virus-induced pathology

Fly lines and <i>Wolbachia</i> strains.

<p>Fly lines and <i>Wolbachia</i> strains.</p

The effect of different <i>Wolbachia</i> strains on the accumulation of FHV in <i>D. simulans.</i>

<p>Graphs show survival of flies infected by <i>w</i>Au (<b>A</b>), <i>w</i>Ri (<b>B</b>), <i>w</i>Ha (<b>C</b>), and <i>w</i>No (<b>D</b>) challenged with FHV (black line) or mock infected (grey line). <i>Wolbachia</i> infected (circle and plus sign) and uninfected (triangle and cross) flies. Error bars represent SEM calculated from three replicates. The survival of FHV infected flies with and without <i>Wolbachia</i> is significantly different for <i>w</i>Au and <i>w</i>Ri (p<0.0001, log rank test on Kaplan-Meier curves). For each fly line a similar result was recorded in a replicate experiment.</p

<i>Wolbachia</i> strain <i>w</i>Mel provides antiviral protection in <i>D. simulans</i>.

<p>(<b>A</b>) Graph shows survival of flies infected with DCV (black line) or mock infected (grey line). <i>w</i>Mel-infected (circle and plus sign) or uninfected (triangle and cross) flies. The survival of DCV infected flies with and without <i>Wolbachia</i> is significantly different (p<0.0001). Error bars represent SEM calculated from three replicate vials. This is a representative experiment which was repeated twice more with similar results. (<b>B</b>) Graph showing accumulation of infectious DCV in <i>w</i>Mel infected (grey bars) or uninfected (white bar) flies. Bars represent means from two replicates with SEM shown, and * indicates a significant difference between the means of day 2 samples (p<0.05, unpaired t test).</p

Relative-density of <i>Wolbachia</i> strains in <i>D. simulans</i>.

<p>For each fly line the graph shows the relative abundance of <i>Wolbachia</i> to host genomic DNA estimated using quantitative PCR. Bars represent the mean of 10 replicates and error bars are SEM.</p

The effect of different <i>Wolbachia</i> strains on the accumulation of DCV in <i>D. simulans</i>.

<p>Graphs show accumulation of infectious DCV in flies with (grey bar) or without (white bar) <i>w</i>Au (<b>A</b>), <i>w</i>Ri (<b>B</b>), <i>w</i>Ha (<b>C</b>), and <i>w</i>No (<b>D</b>). Bars represent means from two replicates with SEM shown, and * indicates a significant difference between the means of day 2 samples (p<0.05, unpaired t test).</p

Antiviral protection of different <i>Wolbachia</i> strains in <i>D. simulans</i>.

<p>Graphs show survival of flies infected by <i>w</i>Au (<b>A</b>), <i>w</i>Ri (<b>B</b>), <i>w</i>Ha (<b>C</b>), and <i>w</i>No (<b>D</b>) challenged with DCV (black line) or mock infected (grey line). Flies with <i>Wolbachia</i> (circle and plus sign) and without <i>Wolbachia</i> (triangle and cross). Error bars represent SEM calculated from three replicates. The survival of DCV infected flies with and without <i>Wolbachia</i> is significantly different for <i>w</i>Au (p<0.0001), <i>w</i>Ri (p<0.0001), and <i>w</i>Ha (p<0.01), using log rank test on Kaplan-Meier curves. Experiments were replicated on at least two additional independent cohorts of flies, and the results for all respective replicates of experiments shown in panel A, B and D were similar, however the replicates for panel C varied (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000656#s2" target="_blank">Results</a>).</p

Variation in Antiviral Protection Mediated by Different Wolbachia Strains in Drosophila simulans

Drosophila C virus (DCV) is a natural pathogen of Drosophila and a useful model for studying antiviral defences. The Drosophila host is also commonly infected with the widespread endosymbiotic bacteria Wolbachia pipientis. When DCV coinfects Wolbachia-infected D. melanogaster, virus particles accumulate more slowly and virus induced mortality is substantially delayed. Considering that Wolbachia is estimated to infect up to two-thirds of all insect species, the observed protective effects of Wolbachia may extend to a range of both beneficial and pest insects, including insects that vector important viral diseases of humans, animals and plants. Currently, Wolbachia-mediated antiviral protection has only been described from a limited number of very closely related strains that infect D. melanogaster. We used D. simulans and its naturally occurring Wolbachia infections to test the generality of the Wolbachia-mediated antiviral protection. We generated paired D. simulans lines either uninfected or infected with five different Wolbachia strains. Each paired fly line was challenged with DCV and Flock House virus. Significant antiviral protection was seen for some but not all of the Wolbachia strain-fly line combinations tested. In some cases, protection from virus-induced mortality was associated with a delay in virus accumulation, but some Wolbachia-infected flies were tolerant to high titres of DCV. The Wolbachia strains that did protect occurred at comparatively high density within the flies and were most closely related to the D. melanogaster Wolbachia strain wMel. These results indicate that Wolbachia-mediated antiviral protection is not ubiquitous, a finding that is important for understanding the distribution of Wolbachia and virus in natural insect populations

Behavioral Deficits at 18-22 Months of Age Are Associated with Early Cerebellar Injury and Cognitive and Language Performance in Children Born Extremely Preterm

To investigate associations in toddlers born extremely preterm (<28 weeks) between neonatal neuroimaging and 18- to 22-month developmental and behavioral outcomes. Cohort analysis from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network Surfactant Positive Airway Pressure and Pulse Oximetry Trial Neuroimaging and Neurodevelopmental Outcomes Study of infants born extremely preterm. Subjects underwent cranial ultrasonography and near-term magnetic resonance imaging (MRI). At 18-22 months of corrected age, the assessment included the Brief Infant Toddler Social Emotional Assessment (BITSEA) Problem and Competence Scale scores and the Bayley Scales of Infant Development, Third Edition (Bayley-III). The BITSEA Problem Scale assesses dysregulation; the Competence Scale assesses social-emotional competence. We examined associations of Problem and Competence scores and positive screen rates with cranial ultrasonography and near-term MRI. Mean BITSEA and Bayley-III scores were compared using ANOVA and positive screen rates with the χ2 test. We computed correlations between BITSEA and Bayley-III scores. Of the 397 children, positive BITSEA screens were found in 34% for the Problem score and 26% for the Competence score. Presence of lesions on near-term MRI that included cerebellar lesions were significantly associated with lower BITSEA Competence but not with Problem scores; Competence scores were inversely related to the presence/significance of lesions. Positive screens on Competence scores and on both Competence and Problem scores were significantly associated with Bayley-III cognitive and language scores <85 (P < .001). Social–emotional competence contributes to deficits in cognitive and language development. Presence of injury on near-term MRI that includes cerebellar lesions is associated with later social–emotional competence and may be a useful predictor to guide early assessment and intervention. ClinicalTrials.gov: NCT00063063 and NCT00233324

Growth Rates of Infants Randomized to Continuous Positive Airway Pressure or Intubation After Extremely Preterm Birth.

Objective To evaluate the effects of early treatment with continuous positive airway pressure (CPAP) on nutritional intake and in-hospital growth rates of extremely preterm (EPT) infants. Study design EPT infants (240/7-276/7 weeks of gestation) enrolled in the Surfactant Positive Airway Pressure and Pulse Oximetry Trial (SUPPORT) were included. EPT infants who died before 36 weeks of postmenstrual age (PMA) were excluded. The growth rates from birth to 36 weeks of PMA and follow-up outcomes at 18-22 months corrected age of EPT infants randomized at birth to either early CPAP (intervention group) or early intubation for surfactant administration (control group) were analyzed. Results Growth data were analyzed for 810 of 1316 infants enrolled in SUPPORT (414 in the intervention group, 396 in the control group). The median gestational age was 26 weeks, and the mean birth weight was 839 g. Baseline characteristics, total nutritional intake, and in-hospital comorbidities were not significantly different between the 2 groups. In a regression model, growth rates between birth and 36 weeks of PMA, as well as growth rates during multiple intervals from birth to day 7, days 7-14, days 14-21, days 21-28, day 28 to 32 weeks PMA, and 32-36 weeks PMA did not differ between treatment groups. Independent of treatment group, higher growth rates from day 21 to day 28 were associated with a lower risk of having a Bayley-III cognitive score Conclusions EPT infants randomized to early CPAP did not have higher in-hospital growth rates than infants randomized to early intubation