Persistence of the 2009 Pandemic Influenza A (H1N1) Virus in Water and on Non-Porous Surface

Knowledge of influenza A virus survival in different environmental conditions is a key element for the implementation of hygiene and personal protection measures by health authorities. As it is dependent on virus isolates even within the same subtype, we studied the survival of the 2009 H1N1 pandemic (H1N1pdm) virus in water and on non-porous surface. The H1N1pdm virus was subjected to various environmental parameters over time and tested for infectivity. In water, at low and medium salinity levels and 4°C, virus survived at least 200 days. Increasing temperature and salinity had a strong negative effect on the survival of the virus which remained infectious no more than 1 day at 35°C and 270 parts per thousand (ppt) of salt. Based on modeled data, the H1N1pdm virus retained its infectivity on smooth non-porous surface for at least 7 days at 35°C and up to 66 days at 4°C. The H1N1pdm virus has thus the ability to persist in water and on glass surface for extended periods of time, even at 35°C. Additional experiments suggest that external viral structures in direct contact with the environment are mostly involved in loss of virus infectivity

Contribution à l'étude des bases moléculaires des virus grippaux à potentiel pandémique dans l'environnement

L'étude de la survie des virus grippaux de type A dans le milieu extérieur est un élémen clé pour une meilleure compréhension de la transmission de ces virus et de leur écologie virale au sens large. Cela pourrait permettre d'anticiper les émergences d'une part, et d'aider les différentes autorités de santé à mettre en place des mesures d'hygiène et de protection individuelle. La survie virale hors de l'hôte peut varier d'un sous-type viral à l'autre et aussi au sein d'un même sous-type. La persistance du pouvoir infectieux au cours du temps a donc été étudiée pour 5 virus : 2 de sous-type H1N1, 1 de sous-type H3N2, et 2 de sous-type H5N1 hautement pathogène (HP). Les virus ont été exposés à plusieurs conditions environnementales et leur infectivité a été mesurée au cours du temps en utilisant la technique de titrage en dilution limite ou Dose Infectieuse en Culture Cellulaire 50% (DICC50). En milieu liquide, jusqu'à trois températures ont été testées : 4, 25 et 35 C, combinées ou non à quatre concentrations en sel (0, 5, 35 et 270 g.L" 1). Pour les souches de sous-type H1N1, la survie virale a également été évaluée sur des surfaces lisses, en utilisant des verres de montre, à 4, 25 ou 35 C. Dans l'eau, à 4 C et sans sel, les virus ont persisté au moins 800 jours. L'augmentation de la température et de la salinité a un effet délétère important sur les virus, qui restent infectieux moins d'une journée à 35 C et 270 g.L"1. Sur surface lisse, les virus restent infectieux au moins 7 jours à 35 C et jusqu'à 66 jours à 4 C. Les virus ont donc la capacité de résister dans l'eau et sur verre de montre pendant de longues périodes, même à 35 C. La quantification du génome viral par RT-PCR quantitative en temps réel et la détection de génome intègre en RT-PCR en point final suggèrent que ce sont principalement les structures virales externes en contact direct avec l'environnement qui seraient impliquées dans la perte d'infectivité. Ces données observationnelles de persistance sont en accord avec les études publiées jusqu'à présent dans la littérature. A ce jour, une seule étude cible les déterminants moléculaires de la persistance des virus dans l'environnement. L'étude des déterminants moléculaires a été conduite en utilisant un modèle de pseudoparticules afin d'éviter le travail dans un laboratoire de niveau de confinement 3 et la production d'un grand nombre de virus mutés par génétique inverse. Les pseudoparticules lentivirales générées exprimaient la HA et la NA et ont été utilisées pour réaliser des cinétiques de survie en milieu liquide. La mise en place de ce système lentiviral a nécessité de nombreuses mises au point expérimentales et a été appliqué au sous-type A(H5N1). Les séquences de la HA et de la NA provenaient de la souche A/Thailand/1(Kan-1)2004 (H5N1). Le rôle potentiel de la N-glycosylation de la HA a été étudié en réalisant des mutations au niveau de sites potentiels de N-glycosylation. Des pseudoparticules mutées pour un seul site de N-glycosylation ont ainsi été produites. Le motif a été rendu non glycosylable en remplaçant l'asparagine (Asn) par une glutamine (Gin). Les résultats préliminaires obtenus suggèrent l'implication de la N-glycosylation dans la persistance observée des virus grippaux. D'autres plasmides doublement et triplement mutés pour la N-glycosylation ont aussi été produits, ainsi que des plasmides mutés au niveau de résidus principalement localisés à l'interface des deux sous-unités de la HA : HA! /HA2 et HA2/HA2. Cependant, l'étude de ces déterminants moléculaires nécessite d'être approfondie et élargie.Knowledge of influenza A virus survival in different environmental conditions is a key element to a better understanding of virus transmission and viral ecology. This study could allow anticipating emergences on the one hand and helping health authorities implementating of hygiene and personal protection measures on the other hand. Virus survival outside the host varies from one viral subtype to the other one and also within a subtype. So, viral infectivity was studied over time for five viruses: 2 of H1N1 subtype, 1 of H3N2 subtype and 2 of highly pathogenic (HP) H5N1 subtype. Viruses were subjected to various environmental parameters over time and tested for infectivity using a microtitre endpoint titration or 50% Tissue Culture Infective Dose (TCID50). In water, up to three temperatures were tested: 4, 25 and 35C, combined or not with four concentrations of sodium chloride (0, 5, 35, 270 part per thousand (ppt)). For H1N1 strains, viral survival was also studied on smooth surface, using watch glasses, at 4, 25 and 35C. In water, at 4C and without sodium chloride, viruses were infectious at least 800 days. Increasing environmental temperature and salinity level had a strong negative effect on the survival of viruses which retained their infectivity no more than 1 day at 35C and 270 ppt of salt. On smooth surface, H1N1 viruses were infectious at least 7 days at 35C and up to 66 days at 4C. The viruses have the ability to persist in water and on glass surface for extended periods of time, even at 35C. The viral genome quantification by quantitative RT-PCR in real time and the detection of the whole M segment by two steps endpoint RT-PCR suggest that external viral structures in direct contact with the environment are mostly involved in virus loss of infectivity. These observational data are in agreement with previously published studies. Until now, only one study focused on the molecular determinants of the résistance of influenza viruses in natural reservoirs and Systems. The study of molecular determinants was performed using a model of lentiviral pseudotypes in order to avoid the work in Biosafety Level 3 (BSL-3) laboratory and the large production of viruses mutated by reverse genetic. Lentiviral pseudotypes produced expressed the HA and the NA glycoproteins at their surface and were used to perform survival kinetics in water. The implementation of this lentiviral System required some experimental improvements and was developed for A(H5N1) viruses. The HA and NA sequences came from the A/Thailand/1 (Kan-1)72004 (H5N1) strain. The potential role of the N-glycosylation of the HA was studied performing mutations in the potential sites of N-glycosylation. Lentiviral pseudotypes mutated for only one glycosylation site were produced. Unsuccessful glycosylation occured changing asparagine (Asn) by glutamine (Gin) in the motif of glycosylation. The preliminary results suggest the involvement of N-glycosylation in the observed persistence of influenza A virus. Plasmids mutated for two or three glycosylation sites and also plasmids mutated for some residues found in the interface of the two HA subunits (HA1/HA2 et HA2/HA2) were produced. Nevertheless, the involvement of these molecular determinants needs further experiments.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Cleavage of hemagglutinin-bearing lentiviral pseudotypes and their use in the study of influenza virus persistence.

Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability

Influenza A virus survival in water is influenced by the origin species of the host cell

International audienceBackground: Influenza A viruses have an envelope made of a lipid bilayer and two surface glycoproteins, the hemagglutinin and the neuraminidase. The structure of the virus is directly dependent on the genetic makeup of the viral genome except the glycosylation moieties and the composition of the lipid bilayer. They both depend on the host cell and are in direct contact with the environment, such as air or water. Virus survival is important for virus transmission from contaminated waters in the case of wild aquatic birds or from contaminated surface or air for humans.Objective: The objective of this study was to check whether the origin species of the host cell has an influence on influenza A virus survival.Method: The persistence in water at 35°C of viruses grown on either mammalian cells or avian cells and belonging to two different subtypes H1N1 and H5N1 was compared.Results: Both H5N1 and H1N1 viruses remained infectious for periods of time as long as 19-25 days, respectively. However, within the same subtype, viruses grown on mammalian cells were more stable in water at 35°C than their counterparts grown on avian cells, even for viruses sharing the same genetic background.Conclusions: This difference in virus stability outside the host is probably connected to the nature of the lipid bilayer taken from the cell or to the carbohydrate side chains of the virus surface glycoproteins. Moreover, the long-lasting survival time might have a critical role in the ecology of influenza viruses, especially for avian viruses

High-density resequencing DNA microarrays in public health emergencies.

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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

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

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

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

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