Identification of human annexin A6 as a novel cellular interactant of influenza A virus M2 protein and regulator of virus budding andrelease

Influenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development. The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells. A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression 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 and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated. In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection.published_or_final_versionPublic HealthDoctoralDoctor of Philosoph

Mesoscale Analysis of Rubber Particle Effect on Indirect Tensile and Flexural Tensile Strength of Crumb Rubber Mortar

This paper presents a mesoscale model to study the influence of rubber particles on the mechanical performance of crumb rubber mortar (CRM). The indirect tensile and flexural behaviors of CRM with different rubber replacement rates, shapes, and sizes were investigated. Rubber mortar is assumed to be a three-phase material composed of rubber aggregate, a mortar matrix, and an interface transition zone (ITZ). Numerical analysis showed that rubber content was the governing factor affecting the reduction rate of indirect tensile and flexural strength. The effect of the ITZ on the tensile strength of CRM was within one percent, which could be ignored. The influence of rubber particle size was investigated by analyzing CRM models containing five different rubber sizes from 0.86 mm to 7 mm. For each size, six different models with randomly distributed rubber particles were set up. CRM models presented a similar average strength even with different rubber particle sizes. However, the strength variation among the random models became higher when the rubber particle size increased. Numerical results also proved that treating rubber particles as pores in modeling led to negligible errors. Then, a prediction formula after considering the increase in air content is provided. Finally, the accuracy of numerical simulations was verified through a series of experimental studies

Utilizing carbon nanotubes in ceramic particle reinforced MMC coatings deposited by laser cladding with Inconel 625 wire

Three types of novel composite coatings were fabricated to simultaneously achieve excellent mechanical properties and high-precision surface appearance by hybridizing carbon nanotubes (CNTs) into reinforcement powder. Results showed a uniformly dispersed morphology of ceramic powders in Inconel 625 composite coatings because CNTs weakened the van der Waals force between adjacent reinforcement powders. The composite coatings presented a sound metallurgical bonding with 316L substrate due to the absence of powder agglomeration. In contrast to Ti(C, N), the decarburization of WC caused the formation of intermetallic compounds dominated by W2C. However, due to the presence of considerable CNTs accompanied by sufficient carbon, more Laves phase and NbC were observed in the clad coatings. In addition, the resultant mechanical properties of these powder mixture reinforced coatings were progressively improved due to the homogeneous microstructures refined by reinforcement particles. The present study offered a reference to simultaneously eliminate the agglomeration of reinforcement powder on a micro-scale and promote the mechanical properties of clad protective coatings

Prediction of Winter Wheat Maturity Dates through Assimilating Remotely Sensed Leaf Area Index into Crop Growth Model

Predicting crop maturity dates is important for improving crop harvest planning and grain quality. The prediction of crop maturity dates by assimilating remote sensing information into crop growth model has not been fully explored. In this study, a data assimilation framework incorporating the leaf area index (LAI) product from Moderate Resolution Imaging Spectroradiometer (MODIS) into a World Food Studies (WOFOST) model was proposed to predict the maturity dates of winter wheat in Henan province, China. Minimization of normalized cost function was used to obtain the input parameters of the WOFOST model. The WOFOST model was run with the re-initialized parameter to forecast the maturity dates of winter wheat grid by grid, and THORPEX Interactive Grand Global Ensemble (TIGGE) was used as forecasting period weather input in the future 15 days (d) for the WOFOST model. The results demonstrated a promising regional maturity date prediction with determination coefficient (R2) of 0.94 and the root mean square error (RMSE) of 1.86 d. The outcomes also showed that the optimal forecasting starting time for Henan was 30 April, corresponding to a stage from anthesis to grain filling. Our study indicated great potential of using data assimilation approaches in winter wheat maturity date prediction

Immunopotentiators Improve the Efficacy of Oil-Emulsion-Inactivated Avian Influenza Vaccine in Chickens, Ducks and Geese.

Combination of CVCVA5 adjuvant and commercial avian influenza (AI) vaccine has been previously demonstrated to provide good protection against different AI viruses in chickens. In this study, we further investigated the protective immunity of CVCVA5-adjuvanted oil-emulsion inactivated AI vaccine in chickens, ducks and geese. Compared to the commercial H5 inactivated vaccine, the H5-CVCVA5 vaccine induced significantly higher titers of hemaglutinin inhibitory antibodies in three lines of broiler chickens and ducks, elongated the antibody persistence periods in geese, elevated the levels of cross serum neutralization antibody against different clade and subclade H5 AI viruses in chicken embryos. High levels of mucosal antibody were detected in chickens injected with the H5 or H9-CVCA5 vaccine. Furthermore, cellular immune response was markedly improved in terms of increasing the serum levels of cytokine interferon-γ and interleukine 4, promoting proliferation of splenocytes and upregulating cytotoxicity activity in both H5- and H9-CVCVA5 vaccinated chickens. Together, these results provide evidence that AI vaccines supplemented with CVCVA5 adjuvant is a promising approach for overcoming the limitation of vaccine strain specificity of protection

The HI antibody titers against H5 or H9 viral antigen in shelf-life test of CVCVA5.

<p>The adjuvants in H5-CVCVA5 were prepared in exclusive water-in-oil form which stored at 4°C for 15-month period, and mixed with the H5 (Re-5) commercial inactivated vaccine (A). The H9-CVCVA5 was prepared as all components of the adjuvants directly addition to the aqueous or oil phase of H9 vaccine during the preparation processes, and preserved for a period of 15-month at 4°C (B). **, <i>P</i> < 0.05.</p

The HI antibody titers against H5 viral antigen in broilers.

<p>White feather broilers (A), yellow feather broilers (B), or dot feather broilers (C). The chickens (n = 20) vaccinated at 10–15 days old, and serum collected at 2-, 3-, and 4-week post-vaccination (wpv). H5-CVCVA5, commercial vaccine (H5-Re5) mixed with CVCVA5 adjuvants. H5, commercial vaccine (H5-Re5). Control, naïve control group. **, <i>P</i> < 0.05. Error bars indicates SEM.</p

The HI antibody titers against H5 viral antigen in domestic geese.

<p>Groups of twenty 14-day-old goslings via subcutaneously route received the prime-boost vaccination of H5 or H5-CVCVA5 vaccines (0.5ml), respectively, and the third group was set as blank control. 2^nd vac., the second vaccination. The horizontal dash line is the qualified antibody titer according to the requirements of commercial vaccine. ***, <i>P</i> < 0.01.</p

Cytotoxicity of CD8⁺ T cells from inbred chickens (B₁₉/B₁₉) vaccinated H5 (Re5) or H9 (NJ02/01) subtype vaccine with or without adjuvants.

<p>Inbred chicken embryo fibroblasts infected with S (H5N1) or NJ02/01 (H9N2) avian influenza viruses were used as target cells. PBMCs derived from the vaccinated inbred chicken or control group were used as effector cell.</p