Development and assessment of a new bioassay for accurate quantification of Type I interferons induced by bovine respiratory viruses.

Type I interferon (IFN-I) plays a major role in antiviral and inflammatory processes of the infected host. In the bovine industry, the bovine respiratory disease complex is a major cause of economic and health problems. This disease is caused by interactions of pathogens, together with environmental and host factors. Several pathogens have been identified as causal agents of respiratory diseases in cattle. To better understand how primary infections by viruses predispose animals to further infections by pathogenic bacteria, tools to accurately detect antiviral and immunoregulatory cytokines are needed. To facilitate the detection and quantification of bovine IFN-I, we have established a new specific and sensitive bioassay studies in the bovine host. This assay is based on a Madin-Darby Bovine Kidney (MDBK) cell line that carries a luciferase gene under the control of the IFN-I inducible bovine Mx1 promoter. Specific luciferase activity was measured after stimulation with serial dilutions of recombinant bovine alpha and beta IFNs and human IFN-α. With this novel bioassay we have successfully measured IFN-I production in supernatant from MDBK cells after stimulation of Toll-like receptors (TLR3, TLR7 and TLR8) and RIG-I-like receptors (RIG-I and MDA5), after viral infection with bovine respiratory pathogens, but also in samples from infected calves. Finally, this new bioassay is an easy-to-use and low cost tool to measure the production of bovine Type-I Interferon.

Energetic cost of building a virus.

Viruses are incapable of autonomous energy production. Although many experimental studies make it clear that viruses are parasitic entities that hijack the molecular resources of the host, a detailed estimate for the energetic cost of viral synthesis is largely lacking. To quantify the energetic cost of viruses to their hosts, we enumerated the costs associated with two very distinct but representative DNA and RNA viruses, namely, T4 and influenza. We found that, for these viruses, translation of viral proteins is the most energetically expensive process. Interestingly, the costs of building a T4 phage and a single influenza virus are nearly the same. Due to influenza's higher burst size, however, the overall cost of a T4 phage infection is only 2-3% of the cost of an influenza infection. The costs of these infections relative to their host's estimated energy budget during the infection reveal that a T4 infection consumes about a third of its host's energy budget, whereas an influenza infection consumes only ≈ 1%. Building on our estimates for T4, we show how the energetic costs of double-stranded DNA phages scale with the capsid size, revealing that the dominant cost of building a virus can switch from translation to genome replication above a critical size. Last, using our predictions for the energetic cost of viruses, we provide estimates for the strengths of selection and genetic drift acting on newly incorporated genetic elements in viral genomes, under conditions of energy limitation.

Virus-like Particle Display of the α-Gal Epitope for the Diagnostic Assessment of Chagas Disease.

The α-Gal antigen [Galα(1,3)Galß(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

A cost-benefit analysis of the physical mechanisms of membrane curvature.

Many cellular membrane-bound structures exhibit distinct curvature that is driven by the physical properties of their lipid and protein constituents. Here we review how cells manipulate and control this curvature in the context of dynamic events such as vesicle-mediated membrane traffic. Lipids and cargo proteins each contribute energy barriers that must be overcome during vesicle formation. In contrast, protein coats and their associated accessory proteins drive membrane bending using a variety of interdependent physical mechanisms. We survey the energy costs and drivers involved in membrane curvature, and draw a contrast between the stochastic contributions of molecular crowding and the deterministic assembly of protein coats. These basic principles also apply to other cellular examples of membrane bending events, including important disease-related problems such as viral egress.

[Bioluminescence in-vivo imaging technology and its application in the study of viral infection--a review].

Bioluminescence imaging is one of the bio-optical imaging techniques which report definite biological event in living animals with genetic modification. With high sensitivity, simple operation and high precision, it is particularly applied in observing vital processes like viral infection and tumor growth in vivo. We summarize the principle of bioluminescence imaging, introduce its application in finding virus replication site, study of interferon (IFN) inhibiting-virus effect and real-time visualization of viral latent infection and reactivation, and preview the trend of bioluminescence imaging technological development.

Modelling the self-assembly of virus capsids.

We use computer simulations to study a model, first proposed by Wales (2005 Phil. Trans. R. Soc. A 363 357), for the reversible and monodisperse self-assembly of simple icosahedral virus capsid structures. The success and efficiency of assembly as a function of thermodynamic and geometric factors can be qualitatively related to the potential energy landscape structure of the assembling system. Even though the model is strongly coarse-grained, it exhibits a number of features also observed in experiments, such as sigmoidal assembly dynamics, hysteresis in capsid formation and numerous kinetic traps. We also investigate the effect of macromolecular crowding on the assembly dynamics. Crowding agents generally reduce capsid yields at optimal conditions for non-crowded assembly, but may increase yields for parameter regimes away from the optimum. Finally, we generalize the model to a larger triangulation number T = 3, and observe assembly dynamics more complex than that seen for the original T = 1 model.

Persistent voids: a new structural metric for membrane fusion.

MOTIVATION: Membrane fusion constitutes a key stage in cellular processes such as synaptic neurotransmission and infection by enveloped viruses. Current experimental assays for fusion have thus far been unable to resolve early fusion events in fine structural detail. We have previously used molecular dynamics simulations to develop mechanistic models of fusion by small lipid vesicles. Here, we introduce a novel structural measurement of vesicle topology and fusion geometry: persistent voids. RESULTS: Persistent voids calculations enable systematic measurement of structural changes in vesicle fusion by assessing fusion stalk widths. They also constitute a generally applicable technique for assessing lipid topological change. We use persistent voids to compute dynamic relationships between hemifusion neck widening and formation of a full fusion pore in our simulation data. We predict that a tightly coordinated process of hemifusion neck expansion and pore formation is responsible for the rapid vesicle fusion mechanism, while isolated enlargement of the hemifusion diaphragm leads to the formation of a metastable hemifused intermediate. These findings suggest that rapid fusion between small vesicles proceeds via a small hemifusion diaphragm rather than a fully expanded one. AVAILABILITY: Software available upon request pending public release. SUPPLEMENTARY INFORMATION: Supplementary data are available on Bioinformatics online.

Monte Carlo simulation of bifurcation in the intracellular viral kinetics.

Intracellular viral kinetics are of special interest because the virion population inside an infected cell is well known to tend to grow exponentially and the corresponding kinetics may be unstable. To clarify the special features of such kinetics, we present Monte Carlo simulations taking into account the key steps of virion formation and competition of the host and viral mRNA for the host translation apparatus. Asymptotically, the model employed predicts either a stable steady state or 'ignition' with the unlimited viral growth. Under steady-state conditions, the mean square fluctuations of the viral genome and virion numbers are found to be appreciably larger than those expected on the basis of the Poissonian distribution. In the case of unstable kinetics, the simulations show the type of deviations from the corresponding mean-field results.

Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products.

High throughput screening, increased accuracy and the coupling of real-time quantitative PCR (Q-PCR) to robotic set-up systems are beginning to revolutionise biotechnology. Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. Methods employed for Q-PCR assay validation as required in ICH Topic Q2A Validation of Analytical Methods: Definitions and Terminology (1st June 1995) are also reviewed.