Application of Super-Resolution and Advanced Quantitative Microscopy to the Spatio-Temporal Analysis of Influenza Virus Replication

With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies

Biomedical Applications of Antiviral Nanohybrid Materials Relating to the COVID-19 Pandemic and Other Viral Crises

The COVID-19 pandemic has driven a global research to uncover novel, effective therapeutical and diagnosis approaches. In addition, control of spread of infection has been targeted through development of preventive tools and measures. In this regard, nanomaterials, particularly, those combining two or even several constituting materials possessing dissimilar physicochemical (or even biological) properties, i.e., nanohybrid materials play a significant role. Nanoparticulate nanohybrids have gained a widespread reputation for prevention of viral crises, thanks to their promising antimicrobial properties as well as their potential to act as a carrier for vaccines. On the other hand, they can perform well as a photo-driven killer for viruses when they release reactive oxygen species (ROS) or photothermally damage the virus membrane. The nanofibers can also play a crucial protective role when integrated into face masks and personal protective equipment, particularly as hybridized with antiviral nanoparticles. In this draft, we review the antiviral nanohybrids that could potentially be applied to control, diagnose, and treat the consequences of COVID-19 pandemic. Considering the short age of this health problem, trivially the relevant technologies are not that many and are handful. Therefore, still progressing, older technologies with antiviral potential are also included and discussed. To conclude, nanohybrid nanomaterials with their high engineering potential and ability to inactivate pathogens including viruses will contribute decisively to the future of nanomedicine tackling the current and future pandemics

Delivery of RNAi Therapeutics to the Airways—From Bench to Bedside

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Microtubules in Influenza Virus Entry and Egress

Influenza viruses are respiratory pathogens that represent a significant threat to public health, despite the large-scale implementation of vaccination programs. It is necessary to understand the detailed and complex interactions between influenza virus and its host cells in order to identify successful strategies for therapeutic intervention. During viral entry, the cellular microenvironment presents invading pathogens with a series of obstacles that must be overcome to infect permissive cells. Influenza hijacks numerous host cell proteins and associated biological pathways during its journey into the cell, responding to environmental cues in order to successfully replicate. The cellular cytoskeleton and its constituent microtubules represent a heavily exploited network during viral infection. Cytoskeletal filaments provide a dynamic scaffold for subcellular viral trafficking, as well as virus-host interactions with cellular machineries that are essential for efficient uncoating, replication, and egress. In addition, influenza virus infection results in structural changes in the microtubule network, which itself has consequences for viral replication. Microtubules, their functional roles in normal cell biology, and their exploitation by influenza viruses will be the focus of this review.ISSN:1999-491

Membrane fusion mediated by the influenza virus hemagglutinin

Der Eintritt von Influenza A Viren in Wirtszellen erfolgt anhand des Hämagglutinin (HA) Proteins. Neueste Entwicklungen zielen darauf ab, die fusionsinduzierende Konformations-änderung des HA und damit die Freisetzung des viralen Genoms in die Wirtszelle zu inhibieren. Der Fusionsprozess ist pH-abhängig da nur bei einem niedrigen pH-Wert (~5.0-6.0) die Protonierung bestimmter Reste innerhalb des HA eine Konformationsänderung, und somit die Membranfusion, auslöst. Die Identifizierung von konservierten, titrierbaren Resten und die Aufklärung der Strukturveränderungen im HA ermöglichen eine gezielte Entwicklung neuer antiviraler Medikamente. In dieser Arbeit wurden bestimmte Histidine im HA mittels umfassender experimenteller und theoretischer Methoden als potentielle pH-Sensoren untersucht. Dabei konnte das Histidin an Position 184 als wichtiger Schalter der pH-induzierten Konformationsänderung identifiziert werden. Außerdem bewirkte der Austausch des geladenen Rests an Position 216 in der Nähe des His184 eine Veränderung der pH-Abhängigkeit des H5 HA aufgrund der Beeinflussung des pKa-Werts des His184. Da die Mutation R216E im HA des hochpathogenen H5N1 Virus in allen Isolaten während der Vogelvirenseuche im Jahr 2003/04 detektiert wurde, deutet das Ergebnis daraufhin, dass diese Mutation zur Entstehung des hochvirulenten Vogelvirus und dessen Adaptierung an den Menschen beigetragen hat. In diesem Zusammenhang wurde auch der Einfluss der pH-Abhängigkeit des HA auf die Fusion und Infektiosität von Viren in lebenden Zellen getestet. Eine destabilisierende Mutation im HA eines rekombinanten WSN-H3 Virus reduzierte dessen Infektions- und Replikationseffizienz in MDCK-Zellen, was auf den endosomalen pH-Wert dieser Zellen zurückgeführt werden konnte. Die Messung der Virus-Endosom-Fusionskinetik in lebenden Zellen machte außerdem die Bedeutung der pH-Abhängigkeit des HA für den Zeitpunkt der Membranfusion und dessen Einfluss auf die Effizienz der Virusinfektion deutlich.The entry of influenza A virus into host cells is established by the hemagglutinin (HA) protein. New antiviral strategies aim to inhibit the fusion inducing conformational change of HA and thereby liberation of the viral genome into the cell. This process is strictly pH dependent since the conformational change of HA initiating the fusion of membranes only occurs upon protonation of yet unknown residues within HA at low pH (~5.0-6.0). The identification of conserved titrable residues and better understanding of the sequential structural rearrangements within HA may facilitate the development of new broad-spectrum antivirals. In the present work His184 and His110 were characterized as potential pH sensors by a comprehensive mutational and computational analysis. The results suggest that His184, but not His110, is an important regulator of HA conformational change at low pH. Furthermore, an exchange of charge at position 216 in vicinity to His184 was shown to alter the pH dependence of conformational change and of fusion in correlation to the known pKa dependence of histidines on neighboring residues. The result advocates that the mutation R216E, which emerged in the highly pathogenic H5 HA in 2003-2004, contributed to an altered acid stability of H5 HA via its effect on His184 and thus to the adaptation of avian H5N1 viruses to the human host. Therefore, the role of an altered acid stability of HA for viral fusion and infectivity in living cells was assessed. Recombinant viruses containing a destabilizing mutation in the HA protein were found to have a reduced infectivity and replication efficiency in MDCK cells compared to the respective wild type. Studying virus-endosome fusion kinetics in these cells we could resolve a significant difference in the timing of fusion induction suggesting that the time-point of fusion is a critical determinant of viral infection efficiency which depends on the endosomal acidification as well as on the acid stability of HA

Determinants Of Hiv-1 Transmission Fitness

DETERMINANTS OF HIV-1 TRANSMISSION FITNESS Shilpa S. Iyer Beatrice H. Hahn HIV-1 is predominantly transmitted by mucosal routes and almost 80 percent of new infections are initiated by a single variant. The elucidation of the biological properties of transmitted viruses which distinguish them from non-transmitted variants are critical for the development of therapeutic interventions. To identify such properties, we characterized the biology of 300 limiting dilution-derived virus isolates from the plasma and genital secretions of eight HIV-1 donor and recipient transmission pairs representing the most prevalent subtypes (B and C). Recipient viruses were more infectious per viral particle as determined on a reporter cell line, replicated to higher titers and were released more efficiently from infected primary CD4+ T cells than the corresponding donor isolates. Recipient viruses were more resistant to the inhibitory effects of IFN-α2 and IFN-β evidenced as higher half-maximal inhibitory concentrations and higher replication at the maximal doses of IFN-α2 and IFN-β than corresponding donor isolates. Interestingly, pretreatment of CD4+ T cells with IFN-β, but not IFN-α2 selected donor plasma isolates that exhibited phenotypes similar to transmitted viruses. This suggests that transmitted variants are distinct and that the selective pressure imposed by type I interferons may in part be responsible for the bottleneck associated with mucosal transmission. We next wanted to assess the role of the interferon stimulated gene, tetherin in the antiviral state established by type I IFNs. Thus, we introduced mutations into the vpu gene of various HIV-1 constructs to specifically disrupt their Vpu-mediated tetherin antagonism, and determined the effect on replication and release from infected cells in the presence and absence of IFN-α2. Mutations at key residues in Vpu reduced the viral particle production and release from infected primary CD4+ T cells and this was particularly evident in IFN-α2-treated cells. Interestingly, transmitted HIV-1 variants were released to higher levels from infected cells than chronic control viruses, even in the absence of Vpu. Thus, the counteraction of tetherin resulting in efficient particle release is an important determinant of the interferon resistance of mucosally transmitted HIV-1