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Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4 + T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

Summary Intraspecific variation in dietary breadth can influence important ecological and evolutionary processes, yet the mechanisms generating this variation are usually unknown. Maternally transmitted bacterial symbionts frequently infect insect herbivores, and many have been shown to mediate key ecological interactions. For polyphagous herbivores, infection with particular symbionts is often strongly correlated with feeding on particular plant species, suggesting that facultative symbionts might directly determine herbivore food plant specificity. However, previous tests of this hypothesis have returned inconsistent results, providing little empirical support for a causal relationship between facultative symbiont infection and dietary breadth. Here, we investigate whether heritable facultative symbionts mediate dietary breadth in the polyphagous aphid, Aphis craccivora. We first determined that asexual clones of the aphid differ dramatically in performance across two leguminous food plants, locust and alfalfa, and could be considered biotypes with distinct ecological characteristics. The heritable symbiont Arsenophonus is strongly associated with locust‐origin aphids. We created experimental lines that share aphid genotypes but differed with respect to Arsenophonus infection status, and compared performance across three food plant species. Naturally Arsenophonus‐infected clones performed 2–4× better on locust and up to 75% worse on two alternate plant species than uninfected controls, clearly demonstrating that Arsenophonus promotes specialization on locust. In both laboratory and field experiments, uninfected locust‐ and alfalfa‐origin clones exhibited similar and modest performance on locust, indicating that the ‘locust‐associated biotype’ would not exist without Arsenophonus. We also hypothesized that moving Arsenophonus, via transinfection, to an alfalfa‐origin lineage would improve performance on locust and serve to expand dietary breadth. Indeed, transinfection doubled aphid performance on locust and halved aphid performance on alfalfa. However, because this aphid lineage naturally performs better on alfalfa, the transinfected symbiont functionally equalized aphid performance between locust and alfalfa, making the alfalfa biotype more generalized. Thus, the same symbiont can either reduce or expand dietary breadth, depending on host genotype. Our results unequivocally demonstrate that symbiont gain or loss can instantaneously and substantially change the topology of food plant use in a polyphagous insect, modifying diet in ways that potentially influence the insect's ecological niche, evolutionary trajectory and pest status. Lay Summary

We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium . Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p = 0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p = 0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 ± 0.932 versus HAZ -1.731 ± 0.984 p = 0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering. Trial Registration: NCT02764918 .

The invasive brown widow spider, Latrodectus geometricus (Araneae: Theridiidae), has spread in multiple locations around the world and, along with it, brought associated organisms such as endosymbionts. We investigated endosymbiont diversity and prevalence across putative native and invasive populations of this spider, predicting lower endosymbiont diversity across the invasive range compared to the native range. First, we characterized the microbial community in the putative native (South Africa) and invasive (Israel and the United States) ranges via high throughput 16S sequencing of 103 adult females. All specimens were dominated by reads from only 1–3 amplicon sequence variants (ASV), and most individuals were infected with an apparently uniform strain of Rhabdochlamydia . We also found Rhabdochlamydia in spider eggs, indicating that it is a maternally-inherited endosymbiont. Relatively few other ASV were detected, but included two variant Rhabdochlamydia strains and several Wolbachia , Spiroplasma and Enterobacteriaceae strains. We then diagnostically screened 118 adult female spiders from native and invasive populations specifically for Rhabdochlamydia and Wolbachia. We found Rhabdochlamydia in 86% of individuals and represented in all populations, which suggests that it is a consistent and potentially important associate of L. geometricus. Wolbachia was found at lower overall prevalence (14%) and was represented in all countries, but not all populations. In addition, we found evidence for geographic variation in endosymbiont prevalence: spiders from Israel were more likely to carry Rhabdochlamydia than those from the US and South Africa, and Wolbachia was geographically clustered in both Israel and South Africa. Characterizing endosymbiont prevalence and diversity is a first step in understanding their function inside the host and may shed light on the process of spread and population variability in cosmopolitan invasive species.

The IMProved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) is evaluating the potential benefit for reduction in major cardiovascular (CV) events from the addition of ezetimibe versus placebo to 40 mg/d of simvastatin therapy in patients who present with acute coronary syndromes and have low-density lipoprotein cholesterol (LDL-C) ≤125 mg/dL. The primary composite end point is CV death, nonfatal myocardial infarction (MI), nonfatal stroke, rehospitalization for unstable angina (UA), and coronary revascularization (≥30 days postrandomization). The simvastatin monotherapy arm’s LDL-C target is <70 mg/dL. Ezetimibe was assumed to further lower LDL-C by 15 mg/dL and produce an estimated ~8% to 9% treatment effect. The targeted number of events is 5,250. We enrolled 18,144 patients with either ST-segment elevation MI (STEMI, n = 5,192) or UA/non–ST-segment elevation MI (UA/NSTEMI, n = 12,952) from October 2005 to July 2010. Western Europe (40%) and North America (38%) were the leading enrolling regions. The STEMI cohort was younger and had a higher percentage of patients naive to lipid-lowering treatment compared with the UA/NSTEMI cohort. The UA/NSTEMI group had a higher prevalence of diabetes, hypertension, and prior MI. Median LDL-C at entry was 100 mg/dL for STEMI and 93 mg/dL for UA/NSTEMI patients. This trial is evaluating LDL-C lowering beyond previously targeted LDL-C levels. The results depend on achieving the desired separation of LDL-C with ezetimibe and on the assumption that ezetimibe’s lowering of LDL-C will have similar event reduction efficacy as the LDL-C lowering from a statin. The results could affect future therapies and guidelines.

Aphids commonly harbor bacterial facultative symbionts that have a variety of effects upon their aphid hosts, including defense against hymenopteran parasitoids and fungal pathogens. The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is infected with the symbiont Arsenophonus sp., which has an unknown role in its aphid host. Our research goals were to document the infection frequency and diversity of the symbiont in field-collected soybean aphids, and to determine whether Arsenophonus is defending soybean aphid against natural enemies. We performed diagnostic PCR and sequenced four Arsenophonus genes in soybean aphids from their native and introduced range to estimate infection frequency and genetic diversity, and found that Arsenophonus infection is highly prevalent and genetically uniform. To evaluate the defensive role of Arsenophonus , we cured two aphid genotypes of their natural Arsenophonus infection through ampicillin microinjection, resulting in infected and uninfected isolines within the same genetic background. These isolines were subjected to parasitoid assays using a recently introduced biological control agent, Binodoxys communis [Braconidae], a naturally recruited parasitoid, Aphelinus certus [Aphelinidae], and a commercially available biological control agent, Aphidius colemani [Braconidae]. We also assayed the effect of the common aphid fungal pathogen, Pandora neoaphidis (Remaudiere & Hennebert) Humber (Entomophthorales: Entomophthoraceae), on the same aphid isolines. We did not find differences in successful parasitism for any of the parasitoid species, nor did we find differences in P. neoaphidis infection between our treatments. Our conclusion is that Arsenophonus does not defend its soybean aphid host against these major parasitoid and fungal natural enemies.

Many arthropod hosts are infected with bacterial endosymbionts that manipulate host reproduction, but few bacterial taxa have been shown to cause such manipulations. Here, we show that a bacterial strain in the genus Rickettsiella causes cytoplasmic incompatibility (CI) between infected and uninfected hosts. We first surveyed the bacterial community of the agricultural spider Mermessus fradeorum (Linyphiidae) using high throughput sequencing and found that individual spiders can be infected with up to five different strains of maternally inherited symbiont from the genera Wolbachia , Rickettsia , and Rickettsiella . The Rickettsiella strain was pervasive, found in all 23 tested spider matrilines. We used antibiotic curing to generate uninfected matrilines that we reciprocally crossed with individuals infected only with Rickettsiella . We found that only 13% of eggs hatched when uninfected females were mated with Rickettsiella -infected males; in contrast, at least 83% of eggs hatched in the other cross types. This is the first documentation of Rickettsiella , or any Gammaproteobacteria, causing CI. We speculate that induction of CI may be much more widespread among maternally inherited bacteria than previously appreciated. Further, our results reinforce the importance of thoroughly characterizing and assessing the inherited microbiome before attributing observed host phenotypes to well-characterized symbionts such as Wolbachia .

Some insects are infected with maternally inherited bacterial endosymbionts that protect them against pathogens or parasitoids. The weevil Sitona obsoletus (=Sitona lepidus) is invasive in New Zealand, and suspected to contain such defensive symbionts, because it is particularly resistant to a Moroccan strain of the parasitoid Microctonus aethiopoides (which successfully attacks many other weevil species), and shows geographic variation in susceptibility to an Irish strain of the same parasitoid. Using 454 pyrosequencing, we investigated the bacterial community associated with S. obsoletus, two other exotic weevils (Sitona discoideus and Listronotus bonariensis) and two endemic New Zealand weevils (Irenimus aequalis and Steriphus variabilis). We found that S. obsoletus was infected by one strain of Wolbachia and two strains of Rickettsia, none of which were found in any other weevil species examined. Using diagnostic PCR, we found that S. obsoletus in the Northland region, where parasitism is highly variable, were primarily infected with Wolbachia and Rickettsia strain 2, indicating that these two symbionts should be investigated for potential defensive properties. In comparison, S. discoideus lacked any apparent maternally inherited bacterial endosymbionts. In the other weevil species, we found a different strain of Wolbachia and two different strains of Spiroplasma. Two weevil species (St. variabilis and L. bonariensis) were infected with distinct strains of Nardonella, the ancestral endosymbiont of weevils, whereas three weevil species (S. obsoletus, S. discoideus, and I. aequalis) lacked evidence for Nardonella infection. However, I. aequalis was consistently infected with a novel Enterobacteriaceae strain, suggesting that a symbiont replacement may have taken place, similar to that described for other weevil clades.

We aimed to determine the efficacy and safety of adding ezetimibe (Ez) to simvastatin (S) in a post–acute coronary syndrome (ACS) population in a prespecified on-treatment analysis. We evaluated 17,706 post-ACS patients from the IMPROVE-IT trial who had low-density lipoprotein cholesterol values between 50 and 125 mg/dL and who received Ez 10 mg/d with S 40 mg/d (Ez/S) or placebo with simvastatin 40 mg/d (P/S). The primary composite end point was cardiovascular death, myocardial infarction, unstable angina, coronary revascularization ≥30 days postrandomization, or stroke. The on-treatment analysis included patients who received study drug for the duration of the trial or experienced a primary end point or noncardiovascular death within 30 days of drug discontinuation. Mean low-density lipoprotein cholesterol values at 1 year were 71 mg/dL for P/S and 54 mg/dL for Ez/S (absolute difference −17 mg/dL = −24%; P < .001). The 7-year Kaplan-Meier estimate of the primary end point occurred in 32.4% in the P/S arm and 29.8% in the Ez/S arm (absolute difference 2.6%; HRadj 0.92 [95% CI 0.87-0.98]; P = .01). The absolute treatment effect favoring Ez/S was 30% greater than in the intention-to-treat analysis of IMPROVE-IT. This analysis provides additional support for the efficacy and safety of adding Ez to S in this high-risk, post-ACS population.