Identification of novel subgroup a variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

<b>BACKGROUND:</b> The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.<p></p> <b>RESULTS:</b> Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.<p></p> <b>CONCLUSIONS:</b> Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge

BACKGROUND: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. PRINCIPAL FINDINGS: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4(+) T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. CONCLUSIONS: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge

Optimal Communication-Computation Tradeoff for Wireless Multimedia Sensor Network Lifetime Maximization

We address the issue of network lifetime maximization for a special class of wireless sensor networks namely, wireless multi-media sensor networks. High data rates, in these networks at the sensor nodes, compared to the conventional sensor networks and the presence of high temporal correlation in the sampled data make them a suitable candidate for the in-network processing, primarily at the sensor node itself. Using these distinguishing features of wireless multi-media sensor networks to our advantage, we have proposed a framework achieving an optimal tradeoff between communication and computation power consumption leading to network lifetime maximization under the delay quality of service constraints. The distributed implementation of the algorithm realizing the proposed framework is achieved using duality theory. A max-min fairness index based measure of network lifetime maximization is studied as a function of end-to-end delay thresholds. Numerical results show how the total network power consumption is distributed between the communication and the computation power consumption components. The results also provide an insight about the maximum and minimum nodal power consumptions. Our results show that the superior performance in terms of max-min fairness index at higher end-to-end delay thresholds is mainly attributed to the relative lower computation cost compared to the communication cost