Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Influenza A Virus Expresses High Levels of an Unusual Class of Small Viral Leader RNAs in Infected Cells

Evidence has recently accumulated suggesting that small noncoding RNAs, and particularly microRNAs, have the potential to strongly affect the replication and pathogenic potential of a range of human virus species. Here, we report the use of deep sequencing to comprehensively analyze small viral RNAs (18 to 27 nucleotides [nt]) produced during infection by influenza A virus. Although influenza A virus differs from most other RNA viruses in that it replicates its genome in the nucleus and is therefore exposed to the nuclear microRNA processing factors Drosha and DGCR8, we did not observe any microRNAs encoded by influenza virus genes. However, influenza virus infection did induce the expression of very high levels—over 100,000 copies per cell by 8 h postinfection—of a population of 18- to 27-nt small viral leader RNAs (leRNAs) that originated from the precise 5′ ends of all eight influenza virus genomic RNA (vRNA) segments. Like the vRNAs themselves, our data indicate that the leRNAs also bear a 5′-terminal triphosphate and are therefore not capable of functioning as microRNAs. Instead, the high-level production of leRNAs may imply a role in another aspect of the viral life cycle, such as regulation of the switch from viral mRNA transcription to genomic RNA synthesis

Rapid Detection and Subtyping of Human Influenza A Viruses and Reassortants by Pyrosequencing

Background: Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. Methodology/Principal Findings: A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. Conclusions/Significance: In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is mor

The transitioning experiences of internationally-educated nurses into a Canadian health care system: A focused ethnography

<p>Abstract</p> <p>Background</p> <p>Beyond well-documented credentialing issues, internationally-educated nurses (IENs) may need considerable support in transitioning into new social and health care environments. This study was undertaken to gain an understanding of transitioning experiences of IENs upon relocation to Canada, while creating policy and practice recommendations applicable globally for improving the quality of transitioning and the retention of IENs.</p> <p>Methods</p> <p>A focused ethnography of newly-recruited IENs was conducted, using individual semi-structured interviews at both one-to-three months (Phase 1) and nine-to-twelve months post-relocation (Phase 2). A purposive sample of IENs was recruited during their orientation at a local college, to a health authority within western Canada which had recruited them for employment throughout the region. The interviews were recorded and transcribed, and data was managed using qualitative analytical software. Data analysis was informed by Roper and Shapira's framework for focused ethnography.</p> <p>Results</p> <p>Twenty three IENs consented to participate in 31 interviews. All IENs which indicated interest during their orientation sessions consented to the interviews, yet 14 did not complete the Phase 2 interview due to reorganization of health services and relocation. The ethno-culturally diverse group had an average age of 36.4 years, were primarily educated to first degree level or higher, and were largely (under) employed as "Graduate Nurses". Many IENs reported negative experiences related to their work contract and overall support upon arrival. There were striking differences in nursing practice and some experiences of perceived discrimination. The primary area of discontentment was the apparent communication breakdown at the recruitment stage with subsequent discrepancy in expected professional role and financial reimbursement.</p> <p>Conclusions</p> <p>Explicit and clear communication is needed between employers and recruitment agencies to avoid employment contract misunderstandings and to enable clear interpretation of the credentialing processes. Pre-arrival orientation of IENs including health care communications should be encouraged and supported by the recruiting institution. Moreover, employers should provide more structured and comprehensive workplace orientation to IENs with consistent preceptorship. Similar to findings of many other studies, diversity should be valued and incorporated into the professional culture by nurse managers.</p

Laparoscopic resection of a residual retroperitoneal tumor mass of nonseminomatous testicular germ cell tumors

Resection of a residual retroperitoneal tumor mass (RRRTM) is standard procedure after combination chemotherapy for metastatic nonseminomatous testicular germ cell tumors (NSTGCT). At the University Medical Center Groningen, 79 consecutive patients with disseminated NSTGCT were treated with cisplatin combination chemotherapy between 2005 and 2007. Laparoscopic RRRTM was performed for patients with RRTM located less than 5 cm ventrally or laterally from the aorta or the vena cava. The 29 patients who fulfilled the criteria had a median age of 25 years (range, 16-59 years). The stages of disease before chemotherapy treatment according to the Royal Marsden classification were 2A (n = 6, 21%), 2B (n = 14, 48%), 2C (n = 3, 10%), and 4 with a lymph node status of N2 (n = 6, 21%). The median duration of laparoscopy was 198 min (range, 122-325 min). The median diameter of the RRTM was 21 mm (range, 11-47 mm). Laparoscopic resection was successful for 25 patients (86%). Conversion was necessary for three patients (10%): two due to bleeding and one because of obesity. One nonplanned hand-assisted procedure (3%) also had to be performed. Histologic examination of the specimens showed fibrosis or necrosis in 12 patients (41%), mature teratoma in 16 patients (55%), and viable tumor in 1 patient (3%). The median hospital stay was 1 day (range, 1-6 days). During a median follow-up period of 47 months (29-70 months), one patient experienced an early relapse (1 month after the end of treatment) (4%). For properly selected patients, laparoscopic resection of RRTM is an improvement in the combined treatment of disseminated NSTGCT and associated with a short hospital stay, minimal morbidity, rapid recovery, and a neat cosmetic result. Long-term data to prove oncologic efficacy are awaited

Cellular Proteins in Influenza Virus Particles

Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes

Concurrent validity of self-rating scale of self-directed learning and self-directed learning instrument among Italian nursing students

BACKGROUND: Self-Directed Learning develops when students take the initiative for their learning, recognising needs, formulating goals, identifying resources, implementing appropriate strategies and evaluating learning outcomes. This should be seen as a collaborative process between the nurse educator and the learner. At the international level, various instruments have been used to measure Self-Directed Learning abilities (SDL), both in original and in culturally-adapted versions. However, few instruments have been subjected to full validation, and no gold standard reference has been established to date. In addition, few researchers have adopted the established tools to assess the concurrent validity of the emerging new tools. Therefore, the aim of this study was to measure the concurrent validity between the Self-Rating Scale of Self-Directed Learning (SRSSDL_Ita) - Italian version and the Self-Directed Learning Instruments (SDLI) in undergraduate nursing students. METHODS: A concurrent validity study design was conducted in a Bachelor level nursing degree programme located in Italy. All nursing students attending the first, second or third year (n=428) were the target sample. The SRSSDL_Ita, and the SDLI were used. The Pearson correlation was used to determine the concurrent validity between the instruments; the confidence of intervals (CI 95%) bias-corrected and accelerated bootstrap (BCa), were also calculated. RESULTS: The majority of participants were students attending their first year (47.9%), and were predominately female (78.5%). Their average age was 22.5\ub14.1. The SDL abilities scores, as measured with the SRSSDL_Ita (min 40, max 200), were, on average, 160.79 (95% CI 159.10-162.57; median 160); while with the SDLI (min 20, max 100), they were on average 82.57 (95% CI 81.79-83.38; median 83). The Pearson correlation between the SRSSDL_Ita and SDLI instruments was 0.815 (CI BCa 95% 0.774-0.848), (p=0.000). CONCLUSIONS: The findings confirm the concurrent validity of the SRSSDL_Ita with the SDLI. The SRSSDL_Ita instrument can be useful in the process of identifying Self-Directed Learning abilities, which are essential for students to achieve the expected learning goals and become lifelong learners

The Critical Role of Notch Ligand Delta-like 1 in the Pathogenesis of Influenza A Virus (H1N1) Infection

Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection

Modelling the progression of pandemic influenza A (H1N1) in Vietnam and the opportunities for reassortment with other influenza viruses

BACKGROUND: A novel variant of influenza A (H1N1) is causing a pandemic and, although the illness is usually mild, there are concerns that its virulence could change through reassortment with other influenza viruses. This is of greater concern in parts of Southeast Asia, where the population density is high, influenza is less seasonal, human-animal contact is common and avian influenza is still endemic. METHODS: We developed an age- and spatially-structured mathematical model in order to estimate the potential impact of pandemic H1N1 in Vietnam and the opportunities for reassortment with animal influenza viruses. The model tracks human infection among domestic animal owners and non-owners and also estimates the numbers of animals may be exposed to infected humans. RESULTS: In the absence of effective interventions, the model predicts that the introduction of pandemic H1N1 will result in an epidemic that spreads to half of Vietnam's provinces within 57 days (interquartile range (IQR): 45-86.5) and peaks 81 days after introduction (IQR: 62.5-121 days). For the current published range of the 2009 H1N1 influenza's basic reproductive number (1.2-3.1), we estimate a median of 410,000 cases among swine owners (IQR: 220,000-670,000) with 460,000 exposed swine (IQR: 260,000-740,000), 350,000 cases among chicken owners (IQR: 170,000-630,000) with 3.7 million exposed chickens (IQR: 1.9 M-6.4 M), and 51,000 cases among duck owners (IQR: 24,000 - 96,000), with 1.2 million exposed ducks (IQR: 0.6 M-2.1 M). The median number of overall human infections in Vietnam for this range of the basic reproductive number is 6.4 million (IQR: 4.4 M-8.0 M). CONCLUSION: It is likely that, in the absence of effective interventions, the introduction of a novel H1N1 into a densely populated country such as Vietnam will result in a widespread epidemic. A large epidemic in a country with intense human-animal interaction and continued co-circulation of other seasonal and avian viruses would provide substantial opportunities for H1N1 to acquire new genes

Intranasal Immunization with Influenza VLPs Incorporating Membrane-Anchored Flagellin Induces Strong Heterosubtypic Protection

We demonstrated previously that the incorporation of a membrane-anchored form of flagellin into influenza virus-like particles (VLPs) improved the immunogenicity of VLPs significantly, inducing partially protective heterosubtypic immunity by intramuscular immunization. Because the efficacy of mucosal vaccination is highly dependent on an adjuvant, and is particularly effective for preventing mucosal infections such as influenza, we determined whether the membrane-anchored flagellin is an efficient adjuvant for VLP vaccines by a mucosal immunization route. We compared the adjuvant effect of membrane-anchored and soluble flagellins for immunization with influenza A/PR8 (H1N1) VLPs by the intranasal route in a mouse model. The results demonstrate that membrane-anchored flagellin is an effective adjuvant for intranasal (IN) immunization, inducing enhanced systemic and mucosal antibody responses. High cellular responses were also observed as shown by cytokine production in splenocyte cultures when stimulated with viral antigens. All mice immunized with flagellin-containing VLPs survived challenge with a high lethal dose of homologous virus as well as a high dose heterosubtypic virus challenge (40 LD50 of A/Philippines/82, H3N2). In contrast, no protection was observed with a standard HA/M1 VLP group upon heterosubtypic challenge. Soluble flagellin exhibited a moderate adjuvant effect when co-administered with VLPs by the mucosal route, as indicated by enhanced systemic and mucosal responses and partial heterosubtypic protection. The membrane-anchored form of flagellin incorporated together with antigen into influenza VLPs is effective as an adjuvant by the mucosal route and unlike standard VLPs, immunization with such chimeric VLPs elicits protective immunity to challenge with a distantly related influenza A virus