Defining Life: The Virus Viewpoint

Are viruses alive? Until very recently, answering this question was often negative and viruses were not considered in discussions on the origin and definition of life. This situation is rapidly changing, following several discoveries that have modified our vision of viruses. It has been recognized that viruses have played (and still play) a major innovative role in the evolution of cellular organisms. New definitions of viruses have been proposed and their position in the universal tree of life is actively discussed. Viruses are no more confused with their virions, but can be viewed as complex living entities that transform the infected cell into a novel organism—the virus—producing virions. I suggest here to define life (an historical process) as the mode of existence of ribosome encoding organisms (cells) and capsid encoding organisms (viruses) and their ancestors. I propose to define an organism as an ensemble of integrated organs (molecular or cellular) producing individuals evolving through natural selection. The origin of life on our planet would correspond to the establishment of the first organism corresponding to this definition

Expression of the gp150 maedi visna virus envelope precursor protein by mammalian expression vectors

There are very few previous reports of expression of native full-length maedi visna virus (MVV) Env gp150 protein in the literature. Therefore the use of different plasmid and viral expression vectors to obtain full-length gp 150 was investigated. A mammalian expression plasmid, pN3-Env, was constructed containing the MVV env gene encoding the precursor protein gp150 Env. The functionality of the recombinant plasmid was tested for expression in HEK293 cells. A recombinant modified vaccinia Ankara virus, MVA-Env, with expression detected in avian cells was also made. The expression of the MVV gp 150 Env precursor protein was shown for the first time upon transfection of the eukaryotic HEK293 cells by the pN3-Env plasmid DNA as demonstrated by Western blot analysis. These plasmid or viral expression vectors are of potential use in MVV vaccines. (c) 2007 Elsevier B.V. All rights reserved

Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus

The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynxassociated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)–DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responseswere generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN- in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV)would induce protectiveimmuneresponses, sheepwere immunized withVMVgag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon- gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduced provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development