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Bernard Moss – One of the best experts on this subject based on the ideXlab platform.

  • elucidating and minimizing the loss by Recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection
    Journal of Virology, 2009
    Co-Authors: Linda S Wyatt, Patricia L Earl, Wei Xiao, Jeffrey L Americo, Catherine A Cotter, Jennifer Vogt, Bernard Moss
    Abstract:

    While characterizing modified vaccinia virus Recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the Recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the Recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary Recombinant vaccines.

  • Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination.
    Nature methods, 2005
    Co-Authors: Arban Domi, Bernard Moss
    Abstract:

    The large capacity of vaccinia virus (VAC) for added DNA, cytoplasmic expression and broad host range make it a popular choice for gene delivery, despite the burdensome need for multiple plaque purifications to isolate Recombinants. Here we describe how a bacterial artificial chromosome (BAC) containing the entire VAC genome can be engineered in Escherichia coli by homologous recombination using bacteriophage lambda-encoded enzymes. The engineered VAC genomes can then be used to produce clonally pure Recombinant viruses in mammalian cells without the need for plaque purification.

  • comparative efficacy of Recombinant modified vaccinia virus ankara expressing simian immunodeficiency virus siv gag pol and or env in macaques challenged with pathogenic siv
    Journal of Virology, 2000
    Co-Authors: Ilnour Ourmanov, Bernard Moss, Linda S Wyatt, Charles R Brown, Miles W Carroll, Liuobov Pletneva, Simoy Goldstein, David Venzon, Vanessa M Hirsch
    Abstract:

    Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env Recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741‐3752, 1996). This MVA-SIV Recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation Recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with Recombinant or nonRecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV Recombinant vaccines as compared with animals that received nonRecombinant MVA (P 5 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student’s t test) and cumulative (P 5 0.010 by log rank test) survival. These results suggest that Recombinant MVA has considerable potential as a vaccine vector for human AIDS.

Antoine Gessain – One of the best experts on this subject based on the ideXlab platform.

  • northern african strains of human t lymphotropic virus type 1 arose from a recombination event
    Retrovirology, 2015
    Co-Authors: Alexandra Desrames, Olivier Cassar, Olivier Gout, Olivier Hermine, Graham P Taylor, Philippe V Afonso, Antoine Gessain
    Abstract:

    Although recombination is a major source of genetic variability in retroviruses, no Recombinant strain had been observed for HTLV-1, the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype A is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, including the complete LTR and a 522-bp fragment of the env gene, which we collected from 19 countries throughout North, West and Central Africa, the continent where the virus has the largest endemic presence. The samples were obtained from 41 HTLV-1 infected individuals with different clinical statuses including ATL, TSP/HAM and asymptomatic carriers. The Recombinant search and breakpoint detection were performed by boot scanning in Simplot to compare inferred clusters of sequences to each other. Finally, molecular clock analyses were performed to date the Recombinant event observed. This led us to demonstrate the presence of Recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a Recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,0 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus (Desrames et al., J. Virology, 88, 2014). In order to precise the geographical distribution of this Recombinant within the African Continent, we studied a new set of samples from HTLV-1 infected patients of diverse African origin. The data on this on-going study will be presented.

  • northern african strains of human t lymphotropic virus type 1 arose from a recombination event
    Journal of Virology, 2014
    Co-Authors: Alexandra Desrames, Olivier Cassar, Olivier Gout, Olivier Hermine, Graham P Taylor, Philippe V Afonso, Antoine Gessain
    Abstract:

    Although recombination is a major source of genetic variability in retroviruses, no Recombinant strain had been observed for human T-lymphotropic virus type 1 (HTLV-1), the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype a is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, which we collected from 19 countries throughout Africa, the continent where the virus has the largest endemic presence. This led us to demonstrate the presence of Recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a Recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,000 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus. IMPORTANCE A number of HTLV-1 subtypes have been described in different populations, but none of the genetic differences between these subtypes have been ascribed to recombination events. Here we report an HTLV-1 Recombinant virus among infected individuals in North Africa. This demonstrates that, contrary to what was thought, recombination can occur and could play a role in the evolution of HTLV-1.

Peter D. Nagy – One of the best experts on this subject based on the ideXlab platform.

  • Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca(2+)/Mn(2+) Pump.
    Journal of Virology, 2019
    Co-Authors: Nikolay Kovalev, Judit Pogany, Peter D. Nagy
    Abstract:

    Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral Recombinants are minus-strand Recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral Recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca(2+)/Mn(2+) pump, regulating intracellular Ca(2+) and Mn(2+) influx into the Golgi apparatus from the cytosol. The interviral Recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral Recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral Recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of Recombinants among related and unrelated viruses. This is the first time that a host factor‘s role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca(2+)/Mn(2+) pump-deficient yeast to search for interviral Recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral Recombinants. Altogether, interviral RNA Recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral Recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca(2+)/Mn(2+) pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

  • interviral recombination between plant insect and fungal rna viruses role of the intracellular ca 2 mn 2 pump
    Journal of Virology, 2019
    Co-Authors: Nikolay Kovalev, Judit Pogany, Peter D. Nagy
    Abstract:

    Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral Recombinants are minus-strand Recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral Recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca(2+)/Mn(2+) pump, regulating intracellular Ca(2+) and Mn(2+) influx into the Golgi apparatus from the cytosol. The interviral Recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral Recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral Recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of Recombinants among related and unrelated viruses. This is the first time that a host factor‘s role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca(2+)/Mn(2+) pump-deficient yeast to search for interviral Recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral Recombinants. Altogether, interviral RNA Recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral Recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca(2+)/Mn(2+) pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.