Analysis of escape variants on their sensitivity to HC33.1-mediated neutralization and binding, and their effect on in vitro viral fitness.

<p>(<b>A</b>) HC33.1 dose-dependent neutralization against viral pool in culture supernatants collected from P0, P18, P27 and P34 with their respective dominant variants bearing the following mutations: N417S, N417T/N434D/K610R, N417T/S419N/N434D/K610R and S395/L413I/N417T was performed by FFU-reduction assay. (<b>B</b>) Dose-dependent neutralization against recombinant HCVcc variants bearing specific mutations, as identified in each phase of viral escape selection was performed by SEAP reporter assay. The IC₅₀ value against each variant is tabulated in the legend. (<b>A, B</b>) Each assay was performed in triplicates and data are shown as percent neutralization, the mean of two experiments ±SD. (<b>C</b>) HC33.1 binding to specific variants, as identified in the legend, by ELISA. Recombinant JFH1 E1E2 wt or the indicated variant E1E2 lysate was captured by GNA in microtiter wells. The wells were then incubated with HC33.1 at the indicated concentrations (0–150 µg/ml). Binding was detected after anti-human IgG-labeled horseradish peroxidase. The <i>y</i>-axis shows the mean optical density values for triplicate wells, the mean of two experiments ±SD. (<b>D</b>). Effect of specific mutations on in vitro viral fitness was determined by measuring virus yield of wt or variant HCVcc bearing the indicated mutations in the focus forming unit assay at an MOI of 0.1. Each assay was performed in triplicates and data are shown as FFU/ml, the mean of two experiments ±SD.</p

Viral evolution in the presence of increasing HMAb HC33.1 concentrations.

<p>(<b>A</b>) Dual antibody immunofluorescence staining of Huh7.5 cells infected with JFH1 2a HCVcc during multiple passages in increasing concentration of HC33.1. IFA is shown for P0, P18, P27 and P31. HCV E2 glycoprotein was stained with HC33.1 under which viral escape variants were selected (green, upper set of panels), or with CBH-5, a neutralizing domain B HMAb that does not share the same epitope with HC33.1 on E2 (green, lower set of panels). Total virus-infected cells were stained with anti-NS3 antibody labeled with Alexa-594 (red). The cells were counterstained with Hoechst nuclear stain H33342 (blue). The captured images were superimposed (merge). (<b>B and C</b>) Sequence analysis of escape variants in increasing concentrations of HC33.1. Circle graph represents the change in composition of variants from selected passages, as indicated on the top, P0–P34. Specific variants are color coded as indicated in the legend. The table presents the corresponding HC33.1 concentration and the relative intensity in IFA binding by HC33.1. The arrow line divides the viral evolution into four phases (Phase I–IV), based on the appearance in each phase of a variant(s) bearing a specific mutation that became dominant. Viral RNA in the corresponding cell culture supernatants were analyzed by single colony sequencing following RT-PCR amplifications.</p

Dose-dependent escape variants and their stability with and without continuing immune pressure.

<p>(<b>A</b>) The composition of the viral pool at P18 containing the dominant variant_{N417T+N434D+K610R} during five additional passages at a constant concentration of 4.5 µg/ml HC33.1. (<b>B</b>) The composition of the viral pool at P27 containing the dominant variant_{N417T+S419N+N434D+K610R} during 15 additional passages after HC33.1 was removed. (<b>C</b>) The composition of the viral pool at P18 containing the dominant variant_{N417T+N434D+K610R} during 15 additional passages after HC33.1 was removed. (<b>D</b>) The composition of the viral pool at P34 containing the dominant variant_{S395P+L413I+N417T} during 8 additional passages after HC33.1 was removed. (<b>A–D</b>) The identified specific variants are color coded as indicated in the legend.</p

Human and mouse antibodies against amino acid 412–423 have different neutralization profiles.

<p>(<b>A</b>) Epitope alignment. Epitopes of three HMAbs: HC33.1; HC33.4 and HC33.8 are compared with murine MAb AP33. Recombinant E1E2 mutant proteins were expressed in 293T cells and cell lysates were analyzed by ELISA. Individual protein expression was normalized by binding of CBH-17, an anti-HCV E2 HMAb to a linear epitope <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Keck1" target="_blank">[25]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Hadlock1" target="_blank">[47]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Keck8" target="_blank">[51]</a>. Red indicates 0–20%, orange 21–40%, brown 41–60%, white 61–100% and green >100% binding, when the residue was replaced by alanine, relative to binding to wt. Dose-dependent neutralization of (<b>B</b>) wt JFH-1 HCVcc, (<b>C</b>) HCVcc variant bearing N417S mutation and (<b>D</b>) bearing N417T mutation were performed by SEAP reporter assay. (<b>B, C, D</b>) Either wt HCVcc or variant HCVcc was incubated with HC33.1 or AP33, at concentrations ranging from 0.1 to 50 µg/ml, prior to infecting the Huh7J-20 cells. Virus infectivity levels were determined by measurement of the SEAP activity released into the medium. (<b>F</b>) Antibody concentration (µg/ml) required to reach 50% neutralization (IC₅₀) for each antibody is summarized. (<b>E</b>) The effect of N417S or N417T mutation on in vitro viral fitness as measured by focus forming assay at an MOI of 0.1. (<b>B–E</b>) Each assay was performed in triplicates and data are shown as percent neutralization, the mean of two experiments ±SD.</p

<i>Phlebotomus papatasi</i> circadian rhythm pathway annotation.

Phlebotomus papatasi circadian rhythm pathway annotation.</p

Chitinase family annotation.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div

Molecular phylogenetic analysis of <i>Lu</i>. <i>longipalpis</i>, <i>P</i>. <i>papatasi</i> and <i>D</i>. <i>melanogaster</i> TRP channel sequences.

The different TRP subfamilies are displayed on the right. The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman +Freq. model with 1000 bootstrap replicates. (TIF)</p

Assembly statistics.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div

BUSCO analysis.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div

Glycosidase Hydrolase family 13 annotation.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div