CYSTUS052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice

Influenza, a respiratory disease caused by influenza viruses, is still a worldwide threat with a high potential to cause a pandemic. Beside vaccination, only two classes of drugs are available for antiviral treatment against the pathogen. Here we show that CYSTUS052, a plant extract from a special variety of Cistus incanus that is rich in polymeric polyphenols, exhibits antiviral activity against a highly pathogenic avian influenza Avirus (H7N7) in cell culture and in a mouse infection model. In vitro and in vivo treatmentwas performed with an aerosol formulation, because the bioavailability of high molecular weight polyphenols is poor. In MDCK cells, a 90% reduction of plaque numbers on cells pre-incubated with the plant extractwas achieved. For in vivo experiments we used a novel monitoring system for influenza A virus-infected mice that allows measurement of body temperature and gross motor-activity of the animals. Mice treated with CYSTUS052 did not develop disease, showed neither differences in their body temperature nor differences in their gross motor-activity and exhibited no histological alterations of the bronchiolus epithelial cells

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.MRC grant: (G0700815), University of Cambridge/Trinity College grant: (Newton Trust), RGC Hong Kong grant: (GRF 768010 M)

Host Genetic Background Strongly Influences the Response to Influenza A Virus Infections

The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD50 to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses

Hochpathogene aviäre Influenza A Viren : Pathogenese, Vakzinierung und antiviraler Wirkstoff

Infektionen des Menschen mit hochpathogenen aviären Influenzaviren (HPAIV) weisen im Vergleich zu den saisonalen Influenza-Fällen einen besonders schweren Krankheitsverlauf auf. Die Sterberate humaner H5N1-Infektionen liegt derzeit bei etwa 60%. Im August 2010 sind nachweislich 504 Personen an vom H5N1-Virus verursachter aviären Influenza erkrankt und 299 daran gestorben. Der Krankheitsverlauf einer HPAIV-Infektion ist durch eine Reduktion der Lymphozyten (Lymphopenie), einer Hyperinduktion von Zytokinen und Chemokinen (Hyperzytokinämie), sowie ein plötzliches akutes Lungen- und Multiorganversagen gekennzeichnet. Die immunologischen und viralen Faktoren, die zu einem solch kritischen Verlauf der H5N1-Infektion führen, sind bislang nicht ausreichend bekannt. Ein weiteres Problem für die Behandlung der HPAIV-Infektion stellt die zunehmende Resistenz der humanpathogenen H5N1-Isolate gegenüber den gängigen antiviralen Medikamenten dar. Auch wenn eine Adaptation des Virus, die eine Weiterverbreitung von Mensch zu Mensch in großen Dimensionen ermöglichen würde, bisher nicht erfolgt ist, lässt sich diese für die Zukunft nicht ausschließen. Die begrenzten Möglichkeiten zur Bekämpfung von HPAIV-Infektionen verdeutlichen den dringenden Bedarf an neuen, effektiven Maßnahmen gegen diese Krankheit. Für die Entwicklung neuer antiviraler Medikamente und Impfungen gegen das Influenza A Virus müssen die Vorgänge, die den kritischen Verlauf der HPAIV-Infektion beeinflussen besser verstanden werden. Das Ziel dieser Dissertation war es, einen besseren Einblick in genau diese Vorgänge zu bekommen und nach alternativen Möglichkeiten zur Behandlung der HPAIV-Infektion zu suchen. Im Rahmen der hier zusammengefassten wissenschaftlichen Publikationen wurde die Rolle des NF-kappaB-Signalweges bei der Bekämpfung viraler Infektionen untersucht. Es folgte eine Studie zur Beteiligung der Hyperzytokinämie an der H5N1-vermittelten Pathogenese. Des Weiteren konnte ein möglicher Mechanismus der die virusvermittelte Lymphopenie verursacht aufgezeigt werden. Weiterhin erfolgte die Charakterisierung immunologischer Mechanismen, die maßgeblich am Schutz gegen HPAIV-Infektionen nach Vakzinierung beteiligt sind. Schließlich, konnte die antivirale Wirkung von Polyphenolen als Alternative zu den gängigen antiviralen Medikamenten in vitro, sowie im Mausmodell gezeigt werden.Infections of humans with the highly pathogenic avian influenza virus (HPAIV) are much more fatal, in contrast to seasonal influenza infections and the current pandemic outbreak. The mortality rate of human HPAIV H5N1 infections is around 60%. In August 2010, 504 confirmed cases of avian H5N1 infections in humans have been reported, while 299 of them were lethal. HPAIV-mediated disease is characterized by lymphopenia, cytokine dysregulation, acute respiratory distress syndrome and multiorgan failure. Virus-host interactions are not only crucial for the defence against influenza virus infections, moreover these sometimes can negatively impact H5N1 mediated disease severity. However, both viral and immunological factors leading to H5N1 mediated severe influenza are only poorly understood. In addition, many of the human H5N1 virus isolates are already resistant to the two main classes of anti-influenza drugs. Therefore, options for control and treatment of H5N1 infections are limited, demonstrating the urgent need for new effective countermeasures against this important disease. Fortunately, the spread of the virus is limited by a rare human-to-human transmission. Although, HPAIV H5N1 has not evolved to a form that allows easy spreading between humans, it is still considered by the World Health Organisation (WHO) as a potential pandemic strain. Moreover, the possibility of a reassortment between the pandemic SOIV H1N1v and H5N1 influenza virus strain is indeed a frightening but feasible association. A mixed strain capable of efficient human-to-human transmission may cause a serious pandemic with fatal mortality rates. A basic requirement for the development of new antiviral agents and vaccines against the influenza A virus is the understanding of how and why influenza viruses cause disease in humans and what influences disease severity. The aim of this Ph.D thesis was to provide a better understanding of the immunological mechanisms that influence the critical outcome of HPAIV infection. The second goal of this work was to identify a new promising anti-influenza agent which can be used against infections with HPAIV. In the range of the scientific publications presented here I investigated the role of the NF-kappaB signalling pathway in viral infections. I determined the impact of two major hallmarks of HPAIV infection, hypercytokinemia and lymphopenia on the critical H5N1-mediated disease outcome. Furthermore, I characterized immune mechanisms that provide cross-protection against lethal influenza A H5N1 virus infection. Finally, as an alternative approach to the common anti-viral drugs I studied the antiviral activity of polyphenols against influenza virus

The alternative NF-kappaB signalling pathway is a prerequisite for an appropriate immune response against lymphocytic choriomeningitis virus infection.

Two major nuclear factor-kappaB (NF-kappaB) signalling pathways are involved in the regulation of the immune response. While the classical NF-kappaB pathway is responsible for regulation of genes encoding components of the innate immune response, the alternative NF-kappaB signalling pathway mediates processes of the adaptive immune system. To evaluate the role of the NF-kappaB signalling pathways in the control of viral infection, we have used lymphocytic choriomeningitis virus (LCMV) infection of mice, which is known to be an excellent model for studying antiviral immune responses. Via the use of mice that were deficient in NF-kappaB subunits from either the classical (p50(-/-) mice) or the alternative NF-kappaB pathway (p52(-/-) mice), we were able to demonstrate that the alternative NF-kappaB pathway is required for the T-cell-mediated immune response against LCMV. Mice that were deficient in the alternative NF-kappaB pathway subunit p52 showed an impaired T-cell response against LCMV infection. Furthermore, these mice also showed an impaired T-cell-dependent humoral immune response against vesicular stomatitis virus (VSV) infection. Adoptive transfer experiments revealed that impaired priming, but not the T-cell response itself, was responsible for the defective cellular immune response against LCMV infection. Our data demonstrate that a functional alternative NF-kappaB signalling pathway is required to assure an adequate immune response after viral infection

Modification of the salivary secretion assay in F508del mice — The murine equivalent of the human sweat test

AbstractBackgroundIn 2005 Best and Quinton established the salivary secretion assay in mice for the in vivo characterization of new drugs against cystic fibrosis (CF). However, limited data are available and the predictive value of this in vivo assay for treatment effects in CF patients is not fully understood.MethodsTherefore, we revisited the salivary secretion assay and systematically investigated the salivary secretion rates in different murine backgrounds and sexes, as well as in different CF mouse models. Moreover, we established quantification of salivary chloride content.ResultsWe found a strain- and sex-dependency of salivary secretion rates and were able to confirm the decreased β-adrenergic salivary secretion response in CFTR knockout mice (CFTRtm1Unc) as well as in the F508del CFTR mice of different origins (CFTRtm1Kth and CFTRtm1Eur). In heterozygous Cftr+/− and Cftr+/F508del mice, the isoprenaline-stimulated salivary secretion rate and the Cl− content were intermediate between values measured in WT and CF mice, indicating that this assay is also able to detect CF carriership. Pilocarpine-induced abnormalities in saliva chloride content in CF mice resembled the changes observed in the human sweat test.ConclusionsDetermination of murine salivary chloride content in combination with salivary secretion rate in CF mice may render the salivary secretion assay as a powerful tool for validation of new CF treatments