70 research outputs found

    Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses

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    <div><p>The <i>Flavivirus</i> genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian/American ZIKV lineage.</p></div

    Positively selected sites in ZIKV polyprotein.

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    <p>Positively selected sites in ZIKV polyprotein.</p

    Branch-site analyses of the flavivirus polyprotein (nonstructural region).

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    <p>Branch-site analyses of the flavivirus polyprotein (nonstructural region).</p

    Ongoing positive selection in ZIKV isolates.

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    <p>(A) Maximum likelihood phylogeny for the nonstructural region (nucleotides 2371 to 8994). Amino acids at the five selected sites are shown for all ZIKV sequences. Viruses isolated from mosquitos and macaques are denoted with hash and asterisk symbols, respectively. Branch length is proportional to nucleotide substitutions per codon. Bootstrap values for internal branches >75% are shown. The phylogenetic tree is unrooted. (B) TMHMM prediction of transmembrane helices (TMH1-5) for the ZIKV NS4B protein and schematic representation of protein topology. Positively selected sites in the flavivirus phylogeny and in ZIKV strains are indicated by red and green triangles, respectively. Amino acid alignments of the regions surrounding selected sites are shown for 5 representative ZIKV, for DENV sequences belonging to the four serotypes, for JEV (NC_001437), and for WNV (strain NY-99, NC_001563). In the alignment, positively selected sites in ZIKV are shown in green; sites that are positively selected in other flaviviruses are marked in magenta. (C) Immune epitope mapping on NS5. Cyan bars indicate the number of epitopes overlapping each NS5 residue. Positively selected sites are colored as above. Mtase: methyltransferase domain; RdRp: RNA-dependent RNA polymerase domain.</p

    Different selective pressures acting on flavivirus polyproteins.

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    <p>(A) Schematic representation of the ZIKV polyprotein. Proteins are colored in hues of blue depending on the percentage of negatively selected sites in ZIKV strains. The location of recombination breakpoints in flaviviruses and ZIKV is shown by striped rectangles. Positively selected sites in the flavivirus phylogeny and in ZIKV strains are colored in red and green, respectively. (B) Maximum likelihood unrooted tree for the flavivirus phylogeny. Branches analyzed in the branch-site tests are indicated with capital letters, with red indicating statistically significant evidence of positive selection. Branch length is proportional to nucleotide substitutions per codon. Bootstrap values for internal branches >75% are shown. See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004978#pntd.0004978.s002" target="_blank">S1 Table</a> for accession number and full names of analyzed viruses. (C) Immune epitope mapping and schematic representation of NS1 domains. Cyan bars indicate the number of epitopes overlapping each NS1 residue. Positively selected sites are also shown.</p

    DataSheet1_Case Report: Decrypting an interchromosomal insertion associated with Marfan’s syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants.pdf

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    Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient’s neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.</p
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