Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

Density‐ and size‐dependent mortality in fish early life stages

The importance of survival and growth variations early in life for population dynamics depends on the degrees of compensatory density dependence and size dependence in survival at later life stages. Quantifying density‐ and size‐dependent mortality at different juvenile stages is therefore important to understand and potentially predict the recruitment to the population. We applied a statistical state‐space modelling approach to analyse time series of abundance and mean body size of larval and juvenile fish. The focus was to identify the importance of abundance and body size for growth and survival through successive larval and juvenile age intervals, and to quantify how the dynamics propagate through the early life to influence recruitment. We thus identified both relevant ages and mechanisms (i.e. density dependence and size dependence in survival and growth) linking recruitment variability to early life dynamics. The analysis was conducted on six economically and ecologically important fish populations from cold temperate and sub‐arctic marine ecosystems. Our results underscore the importance of size for survival early in life. The comparative analysis suggests that size‐dependent mortality and density‐dependent growth frequently occur at a transition from pelagic to demersal habitats, which may be linked to competition for suitable habitat. The generality of this hypothesis warrants testing in future research.publishedVersio

Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC- resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive “Amp-RT” assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings

Characterization of the patterns of drug-resistance mutations in newly diagnosed HIV-1 infected patients naïve to the antiretroviral drugs

<p>Abstract</p> <p>Background</p> <p>The transmission of HIV-1 drug-resistant strains in drug naive patients may seriously compromise the efficacy of a first-line antiretroviral treatment. To better define this problem, a study in a cohort of newly diagnosed HIV-1 infected individuals has been conducted. This study is aimed to assess the prevalence and the patterns of the mutations recently associated with transmitted drug resistance in the reverse transcriptase (RT) and in protease (PR) of HIV-1.</p> <p>Methods</p> <p>Prevalence of transmitted drug resistant strains is determined in 255 newly diagnosed HIV-1 infected patients enrolled in different counselling and testing (CT) centres in Central Italy; the Avidity Index (AI) on the first available serum sample is also used to estimate time since infection. Logistic regression models are used to determine factors associated with infection by drug resistant HIV-1 strains.</p> <p>Results</p> <p>The prevalence of HIV-1 strains with at least one major drug resistance mutation is 5.9% (15/255); moreover, 3.9% (10/255) of patients is infected with HIV nucleoside reverse transcriptase inhibitor (NRTI)-resistant viruses, 3.5% (9/255) with HIV non-NRTI-resistant viruses and 0.4% (1/255) with HIV protease inhibitor (PI)-resistant viruses. Most importantly, almost half (60.0%) of patients carries HIV-1 resistant strains with more than one major drug resistance mutation. In addition, patients who had acquired HIV through homosexual intercourses are more likely to harbour a virus with at least one primary resistance mutation (OR 7.7; 95% CI: 1.7–35.0, P = 0.008).</p> <p>Conclusion</p> <p>The prevalence of drug resistant HIV-1 strains among newly diagnosed individuals in Central Italy is consistent with the data from other European countries. Nevertheless, the presence of drug-resistance HIV-1 mutations in complex patterns highlights an additional potential risk for public health and strongly supports the extension of wide genotyping to newly diagnosed HIV-1 infected patients.</p

From sea monsters to charismatic megafauna: changes in perception and use of large marine animals

Marine megafauna has always elicited contrasting feelings. In the past, large marine animals were often depicted as fantastic mythological creatures and dangerous monsters, while also arousing human curiosity. Marine megafauna has been a valuable resource to exploit, leading to the collapse of populations and local extinctions. In addition, some species have been perceived as competitors of fishers for marine resources and were often actively culled. Since the 1970s, there has been a change in the perception and use of megafauna. The growth of marine tourism, increasingly oriented towards the observation of wildlife, has driven a shift from extractive to non-extractive use, supporting the conservation of at least some species of marine megafauna. In this paper, we review and compare the changes in the perception and use of three megafaunal groups, cetaceans, elasmobranchs and groupers, with a special focus on European cultures. We highlight the main drivers and the timing of these changes, compare different taxonomic groups and species, and highlight the implications for management and conservation. One of the main drivers of the shift in perception, shared by all the three groups of megafauna, has been a general increase in curiosity towards wildlife, stimulated inter alia by documentaries (from the early 1970s onwards), and also promoted by easy access to scuba diving. At the same time, environmental campaigns have been developed to raise public awareness regarding marine wildlife, especially cetaceans, a process greatly facilitated by the rise of Internet and the World Wide Web. Currently, all the three groups (cetaceans, elasmobranchs and groupers) may represent valuable resources for ecotourism. Strikingly, the economic value of live specimens may exceed their value for human consumption. A further change in perception involving all the three groups is related to a growing understanding and appreciation of their key ecological role. The shift from extractive to non-extractive use has the potential for promoting species conservation and local economic growth. However, the change in use may not benefit the original stakeholders (e.g. fishers or whalers) and there may therefore be a case for providing compensation for disadvantaged stakeholders. Moreover, it is increasingly clear that even non-extractive use may have a negative impact on marine megafauna, therefore regulations are needed.SFRH/BPD/102494/2014, UID/MAR/04292/2019, IS1403info:eu-repo/semantics/publishedVersio

Systemic Imidacloprid Affects Intraguild Parasitoids Differently.

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) and Campoletis sonorensis (Cameron) (Hymenoptera, Ichneumonidae) are solitary endoparasitoids of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). They provide biological control of H. virescens populations in Southeastern US agricultural production systems. Field and greenhouse experiments conducted from 2011-2014 compared parasitism rates of parasitoids that developed inside H. virescens larvae fed on tobacco plants treated with and without imidacloprid. The parasitoids in our study did not have a similar response. Toxoneuron nigriceps had reduced parasitism rates, but parasitism rates of C. sonorensis were unaffected. Preliminary data indicate that adult female lifespans of T. nigriceps are also reduced. ELISA was used to measure concentrations of neonicotinoids, imidacloprid and imidacloprid metabolites in H. virescens larvae that fed on imidacloprid-treated plants and in the parasitoids that fed on these larvae. Concentrations were detectable in the whole bodies of parasitized H. virescens larvae, T. nigriceps larvae and T. nigriceps adults, but not in C. sonorensis larvae and adults. These findings suggest that there are effects of imidacloprid on multiple trophic levels, and that insecticide use may differentially affect natural enemies with similar feeding niches

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Special recognition and thanks are extended to the Tennessee Valley Authority for their continued support and financial assistance in publishing these proceedings. Special note is also taken of the assistance and cooperation of Marcia Gray for preparing the proceedings. NCSU Special Bulletin 90-1 July 1990 Conservation Tillage for Agriculture in the 1990’s Proceedings of th

Adult female parasitoids.

<p>Adult females of <i>C</i>. <i>sonorensis</i> (right) and <i>T</i>. <i>nigriceps</i> (left).</p