Variability in Disinfection Resistance between Currently Circulating <i>Enterovirus B</i> Serotypes and Strains

The susceptibility of waterborne viruses to disinfection is known to vary between viruses and even between closely related strains, yet the extent of this variation is not known. Here, different enteroviruses (six strains of coxsackievirus B5, two strains of coxsackievirus B4 and one strain of coxackievirus B1) were isolated from wastewater and inactivated by UV₂₅₄, sunlight, free chlorine (FC), chlorine dioxide (ClO₂), and heat. Inactivation kinetics of these isolates were compared with those of laboratory enterovirus strains (CVB5 Faulkner and echovirus 11 Gregory) and MS2 bacteriophage. FC exhibited the greatest (10-fold) variability in inactivation kinetics between different strains, whereas inactivation by UV₂₅₄ differed only subtly. The variability in inactivation kinetics was greater between serotypes than it was among the seven strains of the CVB5 serotype. MS2 was a conservative surrogate of enterovirus inactivation by UV₂₅₄, sunlight, or heat but frequently underestimated the disinfection requirements for FC and ClO₂. Similarly, laboratory strains did not always reflect the inactivation behavior of the environmental isolates. Overall, there was considerable variability in inactivation kinetics among and within enteroviruses serotypes, as well as between laboratory and environmental isolates. We therefore recommend that future disinfection studies include a variety of serotypes and environmental isolates

The conserved positively charged amino acids in NS4B AH1 are essential for RNA replication

<p>. (<b>A</b>) AH1 mutants introduced into a subgenomic replicon construct harboring a firefly luciferase reporter gene were analyzed in Huh-7.5 cells by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation of <i>in vitro</i> transcribed RNA. Relative light units (RLU) were normalized to values measured at 4 h. ΔGDD represents a replication-deficient control with an inactivating deletion in the RNA-dependent RNA polymerase active site. Results from a representative experiment performed in triplicate are shown. (<b>B</b>) Alanine substitution of the conserved positively charged amino acids in AH1 does not alter the subcellular localization of NS4B. T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different NS4B mutations were transfected into H7-T7-IZ cells which constitutively express the T7 polymerase, followed by immunofluorescence analyses as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#s4" target="_blank">Materials and Methods</a> section. Polyclonal antibody #86 against NS4B and monoclonal antibody 9E10 against NS5A, were used as primary antibodies.</p

Dual membrane topology of HCV NS4B.

<p>The N-terminal part of NS4B assumes a dual membrane topology. Cartoons illustrating the (<b>A</b>) cytosolic and the (<b>B</b>) luminal endoplasmic reticulum (ER) membrane topology of the N-terminal part of NS4B are shown. TMs, predicted transmembrane segments.</p

Analysis of pseudorevertants selected with NS4B AH1 mutant K18A.

<p>(<b>A</b>) Selected amino acid changes identified following transfection of a selectable subgenomic replicon harboring the NS4B AH1 K18A mutation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat-1004501-t001" target="_blank">Table 1</a>) were reengineered into a subgenomic HCV replicon harboring a luciferase reporter gene. <i>In vitro</i> transcribed RNAs were electroporated into Huh-7.5 cells, followed by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation. Relative light units (RLU) were normalized to values measured at 4 h. Results from two independent experiments were pooled (n = 8 for each construct and time point). For statistical analyses, RLU measured at 48 h were compared to the 48-h values obtained with mutant K18A. *, P<0.001. (<b>B</b>) Structure models of the different AH1 sequences were established by using Swiss-PdbViewer 4.01 software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Guex1" target="_blank">[69]</a> and drawn by using VMD 1.9 software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Humphrey1" target="_blank">[68]</a>.</p

The N-terminal part of NS4B assumes a dual topology in a replicative context.

<p>(<b>A</b>) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#s4" target="_blank">Materials and Methods</a> section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#s4" target="_blank">Materials and Methods</a> section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (<b>B</b>) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Schneider1" target="_blank">[70]</a>. (<b>C</b>) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Gouttenoire2" target="_blank">[15]</a>. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).</p

NS4B AH1 is structurally conserved among phylogenetically distant hepaciviruses.

<p>The sequences of NS4B amino acids (aa) 1–70 from the HCV JFH1 strain (genotype 2a; UniProtKB entry Q99IB8) and the non primate hepacivirus strain NPHV-B10-022 (UniProtKB entry I1ZAS8) were aligned by ClustalW (see Legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat-1004501-g001" target="_blank">Figure 1</a> for color code). The similarity index according to ClustalW convention is shown below (asterisk, invariant; colon, highly similar; dot, similar). Helical wheel projections of aa 7–33 of HCV JFH1 and NPHV-B10-022 are shown in the lower panel. The structurally conserved positively charged aa residues flanking the amphipathic α-helices (K, lysine; R, arginine) as well as the glutamate (E) residues on the hydrophilic side are highlighted.</p

Sequence analysis of the N-terminal part of HCV NS4B.

<p>Multiple alignment of NS4B amino acid (aa) 1–70 sequences from representative HCV strains of confirmed genotypes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Simmonds1" target="_blank">[66]</a> are shown (<a href="http://euhcvdb.ibcp.fr" target="_blank">http://euhcvdb.ibcp.fr</a>; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Combet1" target="_blank">[41]</a>). Genotype, GenBank accession number, and strain are indicated for each sequence. Amino acids are numbered with respect to NS4B (top row). The consensus sequence (top row) was deduced from the ClustalW multiple alignment of the indicated NS4B sequences <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Thompson1" target="_blank">[43]</a>. To highlight the aa variability at each position, aa identical to the consensus sequence are indicated by hyphens. The degree of aa physicochemical conservation at each position can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Thompson1" target="_blank">[43]</a> and the consensus hydropathic pattern: o, hydrophobic position (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); n, neutral position (Gly, Ala, Ser, Thr); i, hydrophilic position (Asn, Gln, Asp, Glu, His, Lys, Arg); v, variable position (i.e. when both hydrophobic and hydrophilic residues are observed at a given position). To highlight the variable sequence positions in NS4B, conserved hydrophilic and hydrophobic positions are highlighted in yellow and gray, respectively. Residues are color-coded according to the Wimley and White hydrophobicity scales <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Wimley1" target="_blank">[67]</a>: hydrophobic residues are black (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); polar residues are orange (Gly, Ala, Ser, Thr, Asn and Gln); positively and negatively charged groups of basic (His, Lys, Arg) and acidic residues (Glu, Asp) are blue and red, respectively. Consensus secondary structure predictions of NS4B from representative HCV strains (Second. struct. cons.) are indicated as helical (h, blue) or undetermined (coil [c], orange). Predictions were made by using the web-based algorithms SOPM, HNNC, DSC, GOR IV, PHD, Predator and SIMPA96 available at the NPSA website (<a href="http://npsa-pbil.ibcp.fr" target="_blank">http://npsa-pbil.ibcp.fr</a>; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Combet2" target="_blank">[42]</a> and refs. therein). NMR AH2 structure (bottom row) denotes the conformation of residues determined previously by nuclear magnetic resonance (PDB entry 2JXF; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Gouttenoire4" target="_blank">[26]</a>). Residue conformations are indicated as helical (H) or undetermined (C).</p

Structure of NS4B AH1.

<p>(<b>A</b>) Far UV circular dichroism (CD) spectra of synthetic peptide NS4B[1–40] recorded in 5 mM sodium phosphate pH 7.5 (H₂O), complemented with either 50% 2,2,2-trifluoroethanol (TFE) or 1% L-α-lysophosphatidylcholine (LPC) or the following detergents: 100 mM sodium dodecyl sulfate (SDS), 100 mM n-dodecyl-β-D-maltoside (DDM), or 100 mM dodecylphosphocholine (DPC). (<b>B</b>) Summary of sequential (i, i+1) and medium-range (i, i+2 to i, i+4) nuclear Overhauser enhancements (NOEs) of NS4B[1–40] in 50% TFE. Sequential NOEs allowing the assignment of proline residues are indicated in red. Asterisks indicate that the presence of a NOE cross peak was not confirmed because of overlapping resonances or the lack of H assignment. Intensities of NOEs are indicated by the height of the bars. Amide protons that remained observable in nuclear magnetic resonance (NMR) spectra after three days in 50% D₂0/50% deuterated TFE (TFE-<i>d₃</i>) are indicated by blue squares (slow exchangeable protons). (<b>C</b>) NMR-derived ¹Hα and ¹³Cα chemical shift differences were calculated by subtraction of the experimental values from the reported random coil conformation values in TFE <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Merutka1" target="_blank">[61]</a>. The dashed lines indicate the standard threshold value of ΔHα (−0.1 ppm) or ΔCα (0.7 ppm) for an α-helix. (<b>D</b>) Representative NMR structure of NS4B[1–40] peptide in 50% TFE. The aa sequence of NS4B[1–40] is depicted in (<i>a</i>). The box indicates α-helix residues. Residues are color-coded as described in the Legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat-1004501-g001" target="_blank">Figure 1</a>. (<i>b–d</i>) representative NMR structure model of NS4B[1–40] (PDB entry 2LVG) showing the amphipathic character of α-helix 4–32. <i>b</i>, ribbon representation of the side view colored as in panel <i>a</i>; <i>c</i> and <i>d</i>, hydrophilic side and hydrophobic side views of the molecular surface of amphipathic α-helix 4–32. Figures were generated from structure coordinates using VMD (<a href="http://www.ks.uiuc.edu/Research/vmd/" target="_blank">http://www.ks.uiuc.edu/Research/vmd/</a>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#ppat.1004501-Humphrey1" target="_blank">[68]</a> and rendered with POV-Ray (<a href="http://www.povray.org/" target="_blank">http://www.povray.org/</a>).</p

Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.

<p>(<b>A</b>) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004501#s4" target="_blank">Materials and Methods</a> section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (<b>B</b>) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).</p