Data_Sheet_1_Influenza Virus Segment Composition Influences Viral Stability in the Environment.pdf

<p>The transmission routes of Influenza A viruses (IAVs) submit virus particles to a wide range of environmental conditions that affect their transmission. In water, temperature, salinity, and pH are important factors modulating viral persistence in a strain-dependent manner, and the viral factors driving IAV persistence remain to be described. We used an innovative method based on a real-time cell system analysis to quantify viral decay in an environmental model. Thus, we identified the viral hemagglutinin (HA) and neuraminidase (NA) as the main proteins driving the environmental persistence by comparing the inactivation slopes of several reassortant viruses. We also introduced synonymous and non-synonymous mutations in the HA or in the NA that modulated IAV persistence. Our results demonstrate that HA stability and expression level, as well as calcium-binding sites of the NA protein, are molecular determinants of viral persistence. Finally, IAV particles could not trigger membrane fusion after environmental exposure, stressing the importance of the HA and the NA for environmental persistence.</p

Cleavage of H3 and H1 pseudotypes with TMPRSS2 and HAT proteases.

<p><b>A.</b> Subcellular localization of HA, p24 and TMPRSS2 proteins in transfected HEK 293T cells by structured-illumination fluorescent microscopy. <b>B.</b> Pseudotypes were produced in the presence of trypsin-TPCK or HAT or TMPRSS2, or TMPRSS2 and HAT followed by single-cycle infectivity assays (expressed as the mean log₁₀ RLU ± S.D of triplicate experiments (background limit; 3 log₁₀ RLU). *p<0.01 **p<0.001, and ***p<0.0001 (unpaired Student’s t-test). <b>C.</b> SDS-PAGE and western blot analysis were performed on pseudotypes produced in the presence of TMPRSS2 and HAT, using a mixture of sera specific to H3 and H1 serotypes with HA0 and HA2 representing uncleaved and cleaved HA respectively (Lane 8: H3N2 WIS/05, lane 9: H3N2 VIC/75, lane 10: H1N1 PAR/09, lane 11: H1N1 NC/99). The H3N2 WIS/05 virus was used as a positive control (Lane 1). Uncleaved H3N2 WIS/05 and H1N1 PAR/09 pseudotypes (Lanes 2 and 3 respectively) were used as negative controls. H3N2 WIS/05 and H1N1 PAR/09 pseudotypes were cleaved with either 2 µg.mL⁻¹ of trypsin-TPCK (Lanes 4 and 5 respectively) or with the HAT protease alone (Lanes 6 and 7 respectively).</p

Persistence of H1, H3, and H5 pseudotypes in water.

<p><b>A.</b> P24 capsid ELISA was performed on three independent production of HA bearing pseudotypes in triplicate. PBS and pseudotypes produced in the absence of HA and NA proteins were used as negative controls (PBS and HA-/NA- respectively). <b>B.</b> Quantitative real-time RT-PCR targeting the luciferase genome was performed on three independent production of HA bearing pseudotypes (expressed as the mean Log₁₀ RNA copies.mL⁻¹ ± SD of triplicate repeats). H₂0 and pseudotypes produced in the absence of HA and NA proteins were used as negative controls (H₂0 and HA-/NA- respectively). <b>C.</b> Pseudotypes were diluted in water (1∶2) in the presence (35 g.L⁻¹) or absence (0 g.L⁻¹) of NaCl and were exposed to 4 and 35°C. At each time interval, single-cycle infectivity assays were performed. Loss of infectivity was estimated by calculating the difference between the RLUs after 48 hours and the initial RLU titer. <b>D.</b> Pseudotypes from two independent production stocks were diluted in water (1∶2) in the presence (35 g.L⁻¹) or absence (0 g.L⁻¹) of NaCl and were exposed to 35°C. At each time interval, single-cycle infectivity assays were performed. Loss of infectivity was estimated by calculating the difference between the RLUs after 48 hours and the initial RLU titer. Experiments were carried in triplicates and error bars indicate standard errors of the mean; ***p<0.0001 and NS (not significant) (unpaired Student’s t-test). <b>E.</b> Transmission electronic microcopy of negatively stained H1 pseudotypes, which have been exposed to 35°C and 35 g.L⁻¹ of NaCl for 72 hours.</p

Persistence of H1 pseudotypes in water.

<p>H1N1 pseudotypes were diluted in water (1∶2) in the presence (35 g.L⁻¹) or absence (0 g.L⁻¹) of NaCl and were exposed to 4, 25, and 35°C. At each time interval (represented in hours), single-cycle infectivity assays were performed (represented in mean log₁₀ RLU and background limit; 3 log₁₀ RLU). All experiments were carried out in triplicate. Pseudotypes without HA and NA proteins on their surface (HA-/NA-) were used as a negative control. Error bars indicate standard errors; **p<0.001 and ***p<0.0001 (unpaired Student’s t-test).</p

Cleavage of H3 and H1 pseudotypes with trypsin-TPCK.

<p><b>A.</b> Pseudotypes were exposed to different concentrations of trypsin (0, 2, 100 and 500 µg.mL⁻¹) for 30 min followed by single-cycle infectivity assays and luciferase assays (expressed in Mean log₁₀ Relative Luminescence Units, RLU) with a minimum value of 3.0 log₁₀ RLU, which corresponded to the background limit (grey line). Data are expressed as the mean ± Standard Deviation (S.D) of triplicate repeats. ***p<0.0001 versus 0 µg.mL⁻¹ concentration, NS (not significant), Student's t-test. <b>B.</b> SDS-PAGE/Western blot analysis of pseudotypes bearing different HAs after treatment with trypsin-TPCK. The H3N2 WIS/05 virus was used as a positive control (virus control). Western blot was performed using antibodies targeting the HA protein (HA0 and HA2 representing uncleaved and cleaved HA respectively) or the HIV p24 (loading control). <b>C.</b> Hemolysis assays were performed on different pseudotypes after treatment with 0 or 500 µg.mL⁻¹ trypsin-TPCK. Lysis of guniea pig red blood cells was evaluated by measuring the optical density of released haemoglobin at 540 nm. The H3N2 WIS/05 virus was used as a positive control (virus control) and PBS (phosphate buffered saline) as a negative control. Data are expressed as the mean ± S.D of triplicate repeats. ***p<0.0001, **p<0.001 and NS (not significant), Student's t-test.</p

Production of lentiviral pseudotypes bearing hemagglutinin glycoproteins of influenza A virus and their use in survival kinetics.

<p><b>A.</b> Schematic representation of lentiviral production. 293T cells were co-transfected with plasmids expressing HA and NA, plasmids coding for the lentiviral core (GAG-POL-REV), the encapsidated luciferase genome and HAT/TMPRSS2 proteases. For persistence studies, lentiviral pseudotypes were exposed to water at different temperatures or salinities. At different time intervals, infectivity was assessed through transduction of MDCK cells and luciferase assays. <b>B.</b> Transmission electronic micrographs. Spherical shape of an H1N1 influenza virus (1). Overview of lentiviral pseudotypes with black arrows representing spherical pseudoparticles (2). Negative stained of damaged (3), spherical (4) and pleiomorphic (5) pseudoparticles. Schematic representation of a HA/NA bearing lentiviral pseudotype (6).</p

Sequences of the primers used in RT-PCR for cloning the HA and NA of the different IAV strains.

<p>F: forward primers and R: reverse primers (5′-3′).</p><p>Sequences of the primers used in RT-PCR for cloning the HA and NA of the different IAV strains.</p

MCPyV genome sequence coverage versus MCPyV viral load in the six samples.

<p>Genome coverage is expressed as number of reads per nucleotide and viral load is in genome copies per ng of whole DNA. Coefficient of correlation is: <i>R²</i> = 0.894; and best fit regression equation is: [MCPyV viral load]  = 4.1 [MCPyV coverage]- 28,850.</p

Phylogenic tree of L1 nucleotide sequences.

<p>Bayesian phylogeny (using the Tamura-Nei model TN93 with gamma distribution and invariant sites) of the main alpha, beta, gamma, mu and nu papillomaviruses infecting human inferred from their L1 nucleotide sequences (the list of the taxa included all sequences available from the PAVE database and are available at: <a href="http://pave.niaid.nih.gov/#prototypes?type=human" target="_blank">http://pave.niaid.nih.gov/#prototypes?type=human</a>). The new sequences (red branches) and species reported here belongs to the <i>Gammapapillomavirus</i> genus only, therefore, <i>Alphapapillomavirus</i> and <i>Betapapillomavirus</i> subtrees have been collapsed for clarity reasons. The tree is rooted by a bird <i>Etapapillomavirus</i>: the <i>Fringilla coelebs Papillomavirus</i> (FCPV). The added animal papillomaviruses to the phylogeny are the Roussetus aegyptiacus papillomavirus type 1 (RAP), the Bovine papillomavirus 8 (BPV8) and the Ovine papillomavirus (OPV) type 3. Posterior probabilities are reported for each node and sequences obtained during this study are depicted by grey/red branches. Each Human papillomavirus is noted HPV followed by the number of the species and by its Genbank accession number.</p

Relative proportion of the cutaneous microbiome in each sample.

<p>-*Eukaryotes denotes <i>Fungi</i>, <i>Viridiplantae</i>, <i>Protozoa</i> and <i>Metazoa</i> sequences.</p><p>- **Other denotes metagenome, vectors, RNA virus, endogenous retrovirus and unclassified sequences.</p