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

<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

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

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

Positively selected sites in ZIKV polyprotein.

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

Ongoing positive selection in ZIKV isolates.

<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.

<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

inter-species trees

Trees used in inter-species analyse

inter-species alignments

Alignments used in inter-species analyse

gammaMap_input_files

Input files used for the population genetics-phylogenetics analysi

Particle Identification with the Cherenkov imaging technique using MPGD based Photon Detectors for Physics at COMPASS Experiment at CERN

A novel technology for the detection of single photons has been developed and implemented in 2016 in the Ring Imaging Cherenkov (RICH) detector of the COMPASS Experiment at CERN SPS. Some basic knowledge in the field of particle identification and RICH counters, Micro Pattern Gaseous Detectors (MPGDs) in general and their development for photon detection applications are provided. The characteristics of the COMPASS setup are summarized and the COMPAS RICH-1 detector is described and shown to provide hadron identification in the momentum range between 3 and 55 GeV/c. The THGEM technology is discussed illustrating their characterization as gas multipliers and as reflective photocathodes: large gains and efficient photodetection collections are achieved when using optimized parameters and conditions (hole diameter = THGEM thickness = 0.4 mm; hole pitch = 0.8 mm and no rim; CH4-rich gas mixtures and electric field values > 1 kV/cm at the CsI surface). The intense R\&D program leading to the choice of a hybrid THGEM + Micromegas architecture for the novel detectors is summarized: prototypes construction and test results are presented. The beam test performed at CERN with two 300 mm $\times$ 300 mm active area hybrid prototypes validated this new technology and allowed to demonstrate efficient detection of Cherenkov photons. The optimal design of the detector, consisting in two layers of THGEMs, the first of which is coated with 300 mm thick layer of CsI, couple with a Micromegas on a pad segmented anode with an original design of capacitive – resistive readout is presented. All aspects of the construction, test and assembling from the raw material selection to the procedures applied for the quality assessment are described in detail. The challenges encountered during the detectors assembly, the test in the laboratory and the transportation of the chambers to CERN for the assembly and mounting are then illustrated. The production of the photocathode and the final assembly of the four hybrid detectors, covering an active area of 1.4 m$^2$ are presented. The adventurous installation of the combined Hybrid PDs and Multi Anode Photo Multiplier Tubes with fused silica lenses onto COMPASS RICH-1 and the work for equipping the new detectors with all needed services are presented. The description of the HV control system and of the other services is illustrated and the APV25-based frontend electronics is described together with the studies performed to understand the electronic and physical noises of the new chambers. A preliminary on-line analysis of the detector response and of the performance are presented: an indication that the average number of photons is larger than the neighboring traditional MWPC-based PDs is obtained. The success of the first MPGD-base

GPC, NP and L trees for evolutionary analyses.

GPC, NP and L trees for evolutionary analyses