A nano-view of West Nile virus-induced cellular changes during infection

BACKGROUND: Microscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope's resolution or the invasive nature of the sample preparation procedures. It is also difficult to have a technique that would allow simultaneous imaging of both surface and sub-surface on the same cell. This has hampered endeavours to elucidate virus-host interactions. Atomic Force Microscopy (AFM), which is commonly used in the physical sciences, is now becoming a good correlative form of microscopy used to complement existing optical, confocal and electron microscopy for biological applications RESULTS: In this study, the West Nile (Sarafend) virus-infected Vero cell model was used. The atomic force microscope was found to be useful in producing high resolution images of virus-host events with minimal sample processing requirements. The AFM was able to image the budding of the West Nile (Sarafend) virus at the infected cell surface. Proliferation of the filopodia and thickening of clusters of actin filaments accompanied West Nile virus replication. CONCLUSIONS: The study shows that the AFM is useful for virus-host interaction studies. The technique provides morphological information on both the virus and the host cell during the infection stages

Molecular profiling of T-helper immune genes during dengue virus infection

In this study, we provide a comprehensive molecular profiling of the involvement of T- helper (Th) genes during dengue virus infection of different cell types. The Th gene profiles of three human cell types (monocytes, T-cells and hepatocytes) were analyzed simultaneously via array-based RT-PCR upon infection with dengue virus. Differential regulation of 41 Th genes was identified and of which 20 of those genes may contribute to immuno-pathogenesis of dengue virus infection by regulating inflammation, thrombocytopenia and vascular permeability. Among the strongly up-regulated genes were the RANTES, CC-CKR3, IRF4, CLEC2C, IL-6 and TLR6, which are potent inducer of inflammation and vascular permeability. Profiling genes obtained from this study may serve as potential biomarkers and the modulation of Th immune responses during dengue virus infection has important implications in disease outcome

West Nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the MHC II compartment, and enhanced neutralizing antibody response

AbstractA genetic vaccine for West Nile virus (WN) has been synthesized with the WN premembrane-envelope (WN preM-E) gene sequences encoded as a chimera with the transmembrane and carboxyl terminal domains of the lysosome-associated membrane protein (LAMP). The LAMP sequences are used to direct the antigen protein to the major histocompatibility class II (MHC II) vesicular compartment of transfected professional antigen-presenting cells (APCs). Vaccine constructs encoding the native WN preM-E and WN preM-E/LAMP chimera were synthesized in pVAX1 and pITR plasmid backbones. Extracts of human fibroblast 293 and monkey kidney COS-7 cells transfected with the WN preM-E/LAMP chimera constructs contained much greater amounts of E than did the cells transfected with constructs encoding the native WN preM-E. This difference in the concentration of native E and the E/LAMP chimera in transfected cells is attributed to the secretion of native E. The amount of preM protein in cell extracts, in contrast to the E protein, and the levels of DNA and RNA transcripts, did not differ between WN preM-E- and WN preM-E/LAMP-transfected cells. Additionally, confocal and immunoelectron microscopic analyses of transfected B cells showed localization of the WN preM-E/LAMP chimera in vesicular compartments containing endogenous LAMP, MHC II, and H2-M, whereas native viral preM-E lacking the LAMP sequences was distributed within the cellular vesicular network with little LAMP or MHC II association. Mice immunized with a DNA construct expressing the WN preM-E/LAMP antigen induced significant antibody and long-term neutralization titers in contrast to the minimal and short-lived neutralization titer of mice vaccinated with a plasmid expressing the untargeted antigen. These results underscore the utility of LAMP targeting of the WN envelope to the MHC II compartments in the design of a genetic WN vaccine

Replication of Alphaviruses: A Review on the Entry Process of Alphaviruses into Cells

Alphaviruses are small, enveloped viruses, ~70 nm in diameter, containing a single-stranded, positive-sense, RNA genome. Viruses belonging to this genus are predominantly arthropod-borne viruses, known to cause disease in humans. Their potential threat to human health was most recently exemplified by the 2005 Chikungunya virus outbreak in La Reunion, highlighting the necessity to understand events in the life-cycle of these medically important human pathogens. The replication and propagation of viruses is dependent on entry into permissive cells. Viral entry is initiated by attachment of virions to cells, leading to internalization, and uncoating to release genetic material for replication and propagation. Studies on alphaviruses have revealed entry via a receptor-mediated, endocytic pathway. In this paper, the different stages of alphavirus entry are examined, with examples from Semliki Forest virus, Sindbis virus, Chikungunya virus, and Venezuelan equine encephalitis virus described

Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

<p>Abstract</p> <p>Background</p> <p>Dengue virus (DENV) is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined.</p> <p>Results</p> <p>In this study, we have used a targeted small interfering RNA (siRNA) library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study.</p> <p>Conclusions</p> <p>Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti-viral strategies and treatment of DENV infection.</p

Towards exploratory hypothesis testing and analysis

10.1109/ICDE.2011.5767907Proceedings - International Conference on Data Engineering745-75

Molecular Mechanisms of West Nile Virus Pathogenesis in Brain Cells

We analyzed the response of human glioma cells to West Nile virus infection by investigating host transcriptional changes. Changes in expression of 23 genes showed similarities to those in other neurodegenerative diseases. These changes may be useful as potential biomarkers and elucidate novel mechanisms behind the neuropathology of infection with this virus

The Changeless Core in a Changing Business Environment

Rapid change in business environment poses threat to business survival. It is crucial to examine the core of business sustainability. This paper proposes changeless factors in response to today’s business environment. Specifically, it addresses (1) the people factor (2) the purpose factor and (3) the principle factor as the pillars through which an organization finds its stability in the sea of business change. This paper demonstrates the unchanged factors to ensure business sustainability and to achieve long term objectives. After mapping the conceptual model, the paper devotes particular attention to conceptualizing the changeless cores of business. This research contributes practical insights for managers to further understand the cores of business sustainability. Specifically, it will assist managers to have clear goals in making decisions and managing change to enhance competitiveness in the business environment

Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples