Healthcare providers encounter a wide range of hazards on the job, including exposure to infectious diseases. Protecting them from occupational infectious disease is very important. Healthcare workers use personal protective equipment (PPE) as a measure to decrease the risk of getting infected during patient care. For high-risk diseases like Ebola, Tyvek suits are coverall suits that protect the body and reduce the risk of body fluid exposure. However, a person removing a contaminated suit may also be exposed to virus. Previous studies have shown that enveloped viruses can survive on different types of surfaces, so the objective of this study is to determine the inactivation of bacteriophage Φ6, a surrogate for enveloped human virus, on the surface of Tyvek suits at two different relative humidity levels, 40% and 60% at 22°C. The results showed the inactivation rate of virus was higher at 60% RH than 40% RH. There was ~3log10 (99.9%) reduction of virus inactivation after 6 hours at 40% but ~3log10 (99.9%) inactivation took 9 hours at 60%. This suggests that enveloped viruses can survive on the surface of Tyvek suits for more than 6 hours, and should be considered a potential risk for contamination when they are taken off after use.

Viral transmission from the environment can occur via fomites, but there is uncertainty about which factors most affect viral persistence on fomites. Children are a population highly susceptible to viral infection, and sharing common fomites like toys may spread infection. The objective of this research was to assess the survival of enveloped viruses on the surfaces of children’s toys, using bacteriophage ϕ6 as a surrogate for enveloped human viruses. The survival of infectious ϕ6 virions was observed over a 24 hour period at 22°C and relative humidities of 40% & 60%. On the surface of children’s toys, ϕ6 was better able to persist at 60% RH (log10 reduction< 2 log10) over a 24 hour period than it was at 40% RH (log10 reduction> 6 log10). If ϕ6 virus persists on toy material for up to 24 hours, then viral transmission via shared fomites is certainly significant.

Ebola viruses, and other members of the family filoviridae, are enveloped, negative sense, RNA viruses that can cause hemorrhagic fever. Currently, there are no antivirals or approved vaccines available that target or protect from Ebola virus infection. However, recently, T-cell immunoglobulin and mucin domain-1 (TIM-1) has been identified as an epithelial-cell receptor for filoviruses and could be a potential target for antivirals. However, little is known about how TIM-1 enhances virus entry and the role of TIM-1 during in vivo infection. In order to determine the key residues of TM-1 involved in interaction with virus, we generated a panel of point-mutations in the immunoglobulin-like variable (IgV) domain of TIM-1. We determined that several residues within the IgV domain that are involved in binding of phosphatidylserine (PtdSer) are also critical for Ebola virus entry. Further, we found that TIM-1 interacts with Ebola virus through binding of PtdSer on the viral envelope. PtdSer liposomes, but not phosphatidylcholine liposomes, competed with TIM-1 for EBOV pseudovirion binding and transduction. In addition, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. We also determined that TIM-1 expression can enhance infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped virion associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like particles (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped virus uptake. The PtdSer-binding activity of the IgV domain is essential for both virus binding and internalization by TIM-1. However, another member of the TIM family, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry. These data indicate that other domains of TIM proteins are functionally important. We investigated the domains of the TIM family members that play a role in the enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. Provided that expression of TIM-1 in cells enhances virus entry through binding of PtdSer on the viral membrane, we wanted to determine whether virus entry would still be enhanced if this interaction was reversed with TIM-1 present on the viral membrane. Further, we reasoned that this might allow for targeting of virus to cells with greater amounts of PtdSer exposed on their outer leaflet, such as cancer cells. In order to test this hypothesis, we generated virions in cells coexpressing a glycoprotein and one of the TIM family members. We found that expression of TIMs in virus-producing cells resulted in TIM proteins being released into the virus-containing medium and enhanced Ebola virus GP pseudovirion titers. Further, this enhancement was dependent on the amount of PtdSer exposed on the target-cell membrane. However, we also determined that TIMs were not being incorporated into virions and that coexpression of TIMs with non-ebolavirus glycoproteins in virus-producing cells resulted in virus stocks with both reduced titers and the quantity of virions.

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Physik gibt es überall dort, wo Materie: Maßnahmen wie Energie, Masse, Temperatur, Geschwindigkeit, Größe und Steifigkeit sind alle Beispiele der physikalischen Eigenschaften. Solche Mengen sind wichtige Charakterisierungen für biologische Organismen: Sie verändern die ganze Zeit während des gesamten Lebenszyklus. Für eine Bio-Mechaniker, Steifigkeit ist eine wichtige Maßnahme zur biologischen Design zu verstehen. Weil biologische Bausteine so klein wie 1 nm (Protein / DNA / Lipid) sein können, sind spezielle Techniken erforderlich, um ihre Steifigkeit zu studieren. Beide Rasterkraftmikroskopie (AFM) und optischen Pinzetten können verwendet werden, um aktiv zu verformen die Objekte an pN-nN Kräfte und messen die Verformung auf Nanometer Längenskalen werden. In dieser Arbeit AFM wird angewandt, um die Mechanik von Influenza-Viren, Liposomen und lebenden Zellen zu studieren. Das Genom von Viren von einer Proteinhülle und in einigen Fällen eine zusätzliche Lipidhülle verpackt. Dieser Verbund Shell hat widersprüchliche Rollen: er hat das virale Genom zu schützen, aber es sollte auch ermöglichen Auspacken während der viralen Infektion in das Genom zu lösen. Influenza-Virus ist das weichste Virus jemals gefunden, aber zur gleichen Zeit eine sehr hartnäckige Virus verursacht jährliche Pandemien. Ein besseres Verständnis der mechanischen Eigenschaften des Influenza-Virus kann uns helfen zu verstehen, warum das Virus so erfolgreich ist. Die mechanischen Eigenschaften von Influenza-Viren wurden durch AFM gemessen und mit den Liposomen der viralen Lipid hergestellt. Wir haben gefunden, dass die Influenzavirus-Mechanik durch seine Lipidhülle (~ 70%) werden dominiert. In Kapitel 2 haben wir gezeigt, dass anstelle der Verwendung einer starren Proteinkapsid die Lipidhülle ausreicht, um das Influenza virale Genom zu schützen. In Kapitel 3 haben wir weitere blickte in die Funktion des M1 Proteinhülle während der viralen Infektion. Ein Zwischenprodukt Auspacken Schritt wurde durch Messen der in fluenzavirale Steifigkeit bei pH 7, 6, 5,5 und 5, Bedingungen, die die Ansäuerung Umgebungen auf der viralen Infektion nachahmen Stoffwechselweg entdeckt. Der Zwischenschritt wurde weiterhin als wesentlich erwiesen für eine erfolgreiche Infektion. Wir schlagen vor, dass das Influenza-Virus hat sich zu eng synchronisiert die verschiedenen Schritte ihrer Auspacken mit pH-

Les glycoprotéines de fusion virales de classe 1 contrôlent la fusion entre lenveloppe virale et la membrane cellulaire. Ces glycoprotéines présentent une extrémité N-terminale indispensable à la fusion, le peptide de fusion. Les peptides de fusion sont capables dinduire à eux seuls la fusion de membranes in vitro. Dans cette étude, nous avons dabord analysé les peptides de fusion de gp41 du HIV et de gp30 du BLV. Ces deux peptides de fusion sont des peptides obliques : ils sinsèrent obliquement dans la membrane sous forme hélicoïdale. Nos études ont montré une relation entre la capacité de ces deux peptides de fusion à sinsérer obliquement dans la membrane et la capacité de leurs glycoprotéines de fusion à induire la fusion. Dans le cas du BLV, nous avons également montré une relation entre lobliquité du peptide de fusion et sa fusogénicité. Cette relation obliquité-fusogénicité a été utilisée pour prédire avec succès la région minimale des deux peptides de fusion suffisante pour induire une fusion significative in vitro, qui correspond respectivement aux douze et aux quinze premiers acides aminés de gp41 et gp30. Nos résultats montrent également que le peptide caméléon, un peptide de novo avec une structure labile, sinsère obliquement dans la membrane et induit la fusion in vitro. Le fait que ce peptide fasse partie des peptides obliques, comme les peptides de fusion du HIV et BLV, renforce lhypothèse dun lien entre la fusogénicité des peptides de fusion et leur flexibilité structurale. De nombreuses études réalisées sur les glycoprotéines de fusion de classe 1 indiquent que le domaine transmembranaire intervient également dans la fusion virale. Ce domaine doit être suffisamment long pour que la fusion soit complète. Dans ce travail, nous avons montré quun peptide transmembranaire modèle, le peptide KALR, est capable de sinsérer et dinduire la fusion de liposomes in vitro. En comparant les résultats de modélisation moléculaire avec ceux de FTIR et ceux de la fusion de phase lipidique/perméabilisation de liposomes, nous avons également montré que le taux dinsertion membranaire et la fusogénicité de KALR dépendent de la longueur de son cur hydrophobe. En effet, le taux dinsertion de KALR dans la membrane est beaucoup plus important lorsquil contient un cur hydrophobe lui permettant de traverser entièrement la membrane. Dans cette situation, KALR est capable dinduire la déstabilisation et la fusion de membranes alors que lorsque son cur hydrophobe est trop court pour lui permettre de traverser la membrane, il en est incapable. Ces résultats ont permis dapporter des éléments de compréhension des mécanismes intervenant lors de la fusion induite par les glycoprotéines de fusion virales. / Abstract: Class 1 fusion glycoproteins of viruses are involved in the fusion between viral envelope and cell membrane. The N-terminal extremity of these glycoproteins, called fusion peptide, is essential for fusion. Fusion peptides are able to induce by themselves in vitro membrane fusion. Firstly, we analysed fusion peptides of HIV-1 gp41 and BLV gp30. These two peptides are tilted peptides: they insert obliquely in the membrane when helical. Our studies showed a correlation between the ability of these two fusion peptides to insert obliquely in the membrane and the ability of whole glycoproteins to induce fusion. For BLV, a relationship between the obliquity of the fusion peptide and its fusogenicity was also observed. This obliquity/fusogenicity relationship was used to successfully predict the minimal region of the two fusion peptides sufficient to induce significant in vitro fusion. The minimal fusion peptide corresponds respectively to the twelve and to the fifteen first residues of gp41 and gp30. Our results also showed that the chameleon peptide, a de novo peptide with structural flexibility, inserts obliquely into the membrane and induces in vitro fusion. The fact that this peptide is a tilted peptide, like fusion peptides of HIV-1 and BLV, confirms the hypothesis of a relationship between the fusion peptides fusogenicity and their structural flexibility. A lot of studies on class 1 fusion glycoproteins of viruses indicate that the transmembrane domain is also directly involved in the viral fusion. Glycoproteins must have a domain long enough to induce complete fusion. In this study, we showed that a model transmembrane peptide, KALR peptide, is able to insert into membranes and to induce their fusion. By comparing molecular modelling results with those of FTIR, of liposomes lipid-mixing and of liposomes leakage, we also showed that the insertion rate into the membranes and the fusogenicity of KALR depend on the length of its hydrophobic core. Indeed, the insertion rate of KALR into the membrane is greatly larger when it contains a hydrophobic core long enough to allow the peptide to traverse the membrane. In this situation, KALR is able to destabilize membranes and to induce their fusion, while when it is too short to match the membrane, it is unable to induce fusion. These results allow to better understanding mechanisms involved in the fusion induced by viral fusion glycoproteins.