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  • 1.
    Asghar, Naveed
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Gunaltay, Sezin
    School of Medical Sciences, Örebro University, Örebro, Sweden.
    Tran, Pham Tue Hung
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Höglund, Urban
    Adlego Biomedical AB, Uppsala, Sweden.
    Johansson, Christer
    Academy of Quality Pharm Science and BiQ Pharma AB, Södertälje, Sweden.
    Frelin, Lars
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Sällberg, Matti
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    DNA launched suicidal flaviviruses as therapeutic vaccine candidates2018Konferensbidrag (Refereegranskat)
    Abstract [en]

    Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. The relativelyhigh cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect interventions with high efficiency. We aim to develop therapeutic vaccine candidates against HBV, HCV and HDV using DNA based subgenomic flavivirus replicons as a delivery system. Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), or Kunjinvirus (KUNV) replicon with firefly luciferase geneas a reporter were expressed and characterized in cell culture studies. WNV and KUNV replicons showed significantly higher replication compared to their respective negative controls with unfunctional viral RNA dependent RNA polymerase. KUNV and WNV replicons were chosen for cloning the HCV or HB/DV vaccine candidate gene by replacing luciferasegene. Owing to the self-replicating trait of the flavivirus subgenomic replicons, Western blotting demonstrated that the antigen expression by KUNV and WNV replicons was several folds higher than the positive control. These results suggest that DNA based KUNV and WNV replicons may function as carriers for the hepatitis vaccine candidate genes, and these replicons are currently used for in vivostudies in animal models.

  • 2.
    Asghar, Naveed
    et al.
    School of Natural Science, Technology & Environmental Studies, Södertörn University, Huddinge, Sweden .
    Lee, Yi-Ping
    Department of Clinical Microbiology, Virology,Umeå University, Umeå, Sweden; The Laboratory for Molecular Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
    Nilsson, Emma
    Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; The Laboratory for Molecular Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
    Lindqvist, Richard
    Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; The Laboratory for Molecular Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Kröger, Andrea
    Innate Immunity and Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany; Institute for Microbiology, University of Magdeburg, Magdeburg, Germany.
    Överby, Anna K.
    Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; The Laboratory for Molecular Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikel-id 39265Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The tick-borne encephalitis virus (TBEV) is a flavivirus transmitted to humans, usually via tick bites. The virus causes tick-borne encephalitis (TBE) in humans, and symptoms range from mild flu-like symptoms to severe and long-lasting sequelae, including permanent brain damage. It has been suggested that within the population of viruses transmitted to the mammalian host, quasispecies with neurotropic properties might become dominant in the host resulting in neurological symptoms. We previously demonstrated the existence of TBEV variants with variable poly(A) tracts within a single blood-fed tick. To characterize the role of the poly(A) tract in TBEV replication and virulence, we generated infectious clones of Torö-2003 with the wild-type (A)3C(A)6 sequence (Torö-6A) or with a modified (A)3C(A)38 sequence (Torö-38A). Torö-38A replicated poorly compared to Torö-6A in cell culture, but Torö-38A was more virulent than Torö-6A in a mouse model of TBE. Next-generation sequencing of TBEV genomes after passaging in cell culture and/or mouse brain revealed mutations in specific genomic regions and the presence of quasispecies that might contribute to the observed differences in virulence. These data suggest a role for quasispecies development within the poly(A) tract as a virulence determinant for TBEV in mice.

  • 3.
    Asghar, Naveed
    et al.
    School of Natural Science, Technology & Environmental Studies, Södertörn University, Huddinge, Sweden.
    Lindblom, Pontus
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Melik, Wessam
    School of Natural Science, Technology & Environmental Studies, Södertörn University, Huddinge, Sweden.
    Lindqvist, Richard
    Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden.
    Haglund, Mats
    Department of Infectious Diseases, County Hospital, Kalmar, Sweden.
    Forsberg, Pia
    Division of Infectious Diseases, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Clinic of Infectious Diseases, Linköping University Hospital, Linköping, Sweden.
    Överby, Anna K.
    Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden.
    Andreassen, Åshild
    Division of Infectious Disease Control, Department of Virology, Norwegian Institute of Public Health, Oslo, Norway.
    Lindgren, Per-Eric
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Division of Medical Services, Department of Microbiology, County Hospital Ryhov, Jönköping, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för läkarutbildning. School of Natural Science, Technology & Environmental Studies, Södertörn University, Huddinge, Sweden; RiSC - Inflammatory Response and Infection Susceptibility Centre, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Tick-Borne Encephalitis Virus Sequenced Directly from Questing and Blood-Feeding Ticks Reveals Quasispecies Variance2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 7, artikel-id e103264Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The increased distribution of the tick-borne encephalitis virus (TBEV) in Scandinavia highlights the importance of characterizing novel sequences within the natural foci. In this study, two TBEV strains: the Norwegian Mandal 2009 (questing nymphs pool) and the Swedish Saringe 2009 (blood-fed nymph) were sequenced and phylogenetically characterized. Interestingly, the sequence of Mandal 2009 revealed the shorter form of the TBEV genome, similar to the highly virulent Hypr strain, within the 3' non-coding region (3'NCR). A different genomic structure was found in the 3'NCR of Saringe 2009, as in-depth analysis demonstrated TBEV variants with different lengths within the poly(A) tract. This shows that TBEV quasispecies exists in nature and indicates a putative shift in the quasispecies pool when the virus switches between invertebrate and vertebrate environments. This prompted us to further sequence and analyze the 3'NCRs of additional Scandinavian TBEV strains and control strains, Hypr and Neudoerfl. Toro 2003 and Habo 2011 contained mainly a short (A) 3C(A)6 poly(A) tract. A similar pattern was observed for the human TBEV isolates 1993/783 and 1991/4944; however, one clone of 1991/4944 contained an (A) 3C(A)11 poly(A) sequence, demonstrating that quasispecies with longer poly(A) could be present in human isolates. Neudoerfl has previously been reported to contain a poly(A) region, but to our surprise the resequenced genome contained two major quasispecies variants, both lacking the poly(A) tract. We speculate that the observed differences are important factors for the understanding of virulence, spread, and control of the TBEV.

  • 4.
    Asghar, Naveed
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Maravelia, Panagiota
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Caro-Perez, Noelia
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Tarn, Hung
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Pasetto, Anna
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Ahlen, Gustaf
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Frelin, Lars
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Höglund, Urban
    Johansson, Christer
    Sällberg, Matti
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses2019Konferensbidrag (Refereegranskat)
    Abstract [en]

    Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. Chronic infections with the hepatitis C virus (HCV) can be effectively cured by antivirals. However, as cured patients can be re-infected they lack protective immune responses. In addition, the relativelyhigh cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect vaccines with high efficiency to control and possibly eradicate Hepatitis viruses globally. The vaccine should induce either, or both, neutralizing antibodies and protective T cell responses. We therefore have developed DNA based flavivirus replicons as a potent delivery system that effectively prime HCV-specific T cell responses. We generated suicidal subgenomic DNA replicons of Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), and Kunjinvirus (KUNV) expressing either a fusion protein between the HCV NS3/4A and a stork hepatitis B virus core or a vaccine candidate gene of HB/DV. Transfection experiments showed that the antigen expression by KUNV and WNV replicons was several folds higher than the antigen expression by standard DNA plasmid with CMV promoter. The immunogenicity of three suicidal flaviviral DNA replicons expressing HCV NS3/4A was tested in mice and compared to HCV NS3/4A expression by the standard DNA plasmid. The KUNV-HCV replicon was the best replicon-based immunogen with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, dextramer staining, and polyfunctionality. Importantly, a mutant KUNV-HCV immunogen lacking replication failed to induce immune responses. Thus, the newly developed KUNV-based suicidal DNA launched replicon vaccine for HCV is a highly attractive candidate as a prophylactic vaccine against chronic hepatitis C. In addition, we are currently testing the immunogenicity of KUNV-HB/DV replicon in mice.

  • 5.
    Asghar, Naveed
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Maravelia, Panagiota
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Caro-Perez, Noelia
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Tran, Pham Tue Hung
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Pasetto, Anna
    aboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Ahlen, Gustaf
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Frelin, Lars
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Höglund, Urban
    Adlego Biomedical AB, Uppsala, Sweden.
    Johansson, Christer
    Academy of Quality Pharm Science and BiQ Pharma AB, Södertälje, Sweden.
    Sällberg, Matti
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses2019Konferensbidrag (Refereegranskat)
    Abstract [en]

    Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. Chronic infections with the hepatitis C virus (HCV) can be effectively cured by antivirals. However, as cured patients can be re-infected they lack protective immune responses. In addition, the relativelyhigh cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect vaccines with high efficiency to control and possibly eradicate Hepatitis viruses globally. The vaccine should induce either, or both, neutralizing antibodies and protective T cell responses. We therefore have developed DNA based flavivirus replicons as a potent delivery system that effectively prime HCV-specific T cell responses. We generated suicidal subgenomic DNA replicons of Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), and Kunjinvirus (KUNV) expressing either a fusion protein between the HCV NS3/4A and a stork hepatitis B virus core or a vaccine candidate gene of HB/DV. Transfection experiments showed that the antigen expression by KUNV and WNV replicons was several folds higher than the antigen expression by standard DNA plasmid with CMV promoter. The immunogenicity of three suicidal flaviviral DNA replicons expressing HCV NS3/4A was tested in mice and compared to HCV NS3/4A expression by the standard DNA plasmid. The KUNV-HCV replicon was the best replicon-based immunogen with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, dextramer staining, and polyfunctionality. Importantly, a mutant KUNV-HCV immunogen lacking replication failed to induce immune responses. Thus, the newly developed KUNV-based suicidal DNA launched replicon vaccine for HCV is a highly attractive candidate as a prophylactic vaccine against chronic hepatitis C. In addition, we are currently testing the immunogenicity of KUNV-HB/DV replicon in mice.

  • 6.
    Bertrand, Yann
    et al.
    Dept Plant & Environm Sci, Univ Gothenburg, Gothenburg, Sweden.
    Töpel, Mats
    Dept Plant & Environm Sci, Univ Gothenburg, Gothenburg, Sweden.
    Elväng, Annelie
    Sch Life Sci, Södertörn Univ, Huddinge, Sweden.
    Melik, Wessam
    Sch Life Sci, Södertörn Univ, Huddinge, Sweden; Dept Genet Microbiol & Toxicol, Stockholm Univ, Stockholm, Sweden.
    Johansson, Magnus
    Sch Life Sci, Södertörn Univ, Huddinge, Sweden.
    First dating of a recombination event in mammalian tick-borne flaviviruses2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 2, artikel-id e31981Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

  • 7.
    Elväng, Annelie
    et al.
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Melik, Wessam
    School of Life Sciences, Södertörn University, Huddinge, Sweden; Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.
    Bertrand, Yann
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Lönn, Mikael
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Johansson, Magnus
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Sequencing of a tick-borne encephalitis virus from Ixodes ricinus reveals a thermosensitive RNA switch significant for virus propagation in ectothermic arthropods2011Ingår i: Vector Borne and Zoonotic Diseases, ISSN 1530-3667, E-ISSN 1557-7759, Vol. 11, nr 6, s. 649-658Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tick-borne encephalitis virus (TBEV) is a flavivirus with major impact on global health. The geographical TBEV distribution is expanding, thus making it pivotal to further characterize the natural virus populations. In this study, we completed the earlier partial sequencing of a TBEV pulled out of a pool of RNA extracted from 115 ticks collected on Torö in the Stockholm archipelago. The total RNA was sufficient for all sequencing of a TBEV genome (Torö-2003), without conventional enrichment procedures such as cell culturing or suckling mice amplification. To our knowledge, this is the first time that the genome of TBEV has been sequenced directly from an arthropod reservoir. The Torö-2003 sequence has been characterized and compared with other TBE viruses. In silico analyses of secondary RNA structures formed by the two untranslated regions revealed a temperature-sensitive structural shift between a closed replicative form and an open AUG accessible form, analogous to a recently described bacterial thermoswitch. Additionally, novel phylogenetic conserved structures were identified in the variable part of the 3'-untranslated region, and their sequence and structure similarity when compared with earlier identified structures suggests an enhancing function on virus replication and translation. We propose that the thermo-switch mechanism may explain the low TBEV prevalence often observed in environmentally sampled ticks. Finally, we were able to detect variations that help in the understanding of virus adaptations to varied environmental temperatures and mammalian hosts through a comparative approach that compares RNA folding dynamics between strains with different mammalian cell passage histories.

  • 8.
    Johansson, Magnus
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Frelin, Lars
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Maravelia, Panagiota
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Asghar, Naveed
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Caro-Perez, Noelia
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Pasetto, Anna
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Ahlen, Gustaf
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Sallberg, Matti
    Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Immunogenicity of a New Flaviviral-Based DNA Launched Suicidal Replicon for Protective Vaccination Against Hepatitis C2019Ingår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 27, nr 4, s. 139-139Artikel i tidskrift (Övrigt vetenskapligt)
  • 9.
    Kellman, Eliza M.
    et al.
    Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton MT, USA.
    Offerdahl, Danielle K.
    Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton MT, USA.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Bloom, Marshall E.
    Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton MT, USA.
    Viral Determinants of Virulence in Tick-Borne Flaviviruses2018Ingår i: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 10, nr 6, artikel-id 329Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Tick-borne flaviviruses have a global distribution and cause significant human disease, including encephalitis and hemorrhagic fever, and often result in neurologic sequelae. There are two distinct properties that determine the neuropathogenesis of a virus. The ability to invade the central nervous system (CNS) is referred to as the neuroinvasiveness of the agent, while the ability to infect and damage cells within the CNS is referred to as its neurovirulence. Examination of laboratory variants, cDNA clones, natural isolates with varying pathogenicity, and virally encoded immune evasion strategies have contributed extensively to our understanding of these properties. Here we will review the major viral determinants of virulence that contribute to pathogenesis and influence both neuroinvasiveness and neurovirulence properties of tick-borne flaviviruses, focusing particularly on the envelope protein (E), nonstructural protein 5 (NS5), and the 3 untranslated region (UTR).

  • 10.
    Melik, Wessam
    et al.
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Ellencrona, Karin
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Wigerius, Michael
    School of Life Sciences, Södertörn University, Huddinge, Sweden; Department of Pharmacology, Faculty of Medicine, Dalhousie University, Halifax NS, Canada.
    Hedström, Christer
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Elväng, Annelie
    School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för läkarutbildning. School of Life Sciences, Södertörn University, Huddinge, Sweden.
    Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication2012Ingår i: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 169, nr 1, s. 54-62Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The flavivirus genus includes important human neurotropic pathogens like Tick-borne encephalitis virus (TBEV) and West-Nile virus (WNV). Flavivirus replication occurs at replication complexes, where the NS5 protein provides both RNA cap methyltransferase and RNA-dependent RNA polymerase activities. TBEVNS5 contains two PDZ binding motifs (PBMs) important for specific targeting of human PDZ proteins including Scribble, an association important for viral down regulation of cellular defense systems and neurite outgrowth. To determine whether the PBMs of TBEVNS5 affects virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The PBMs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence TBEV replication. We also find that the C-terminal PBMs present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has affinity to Zonula occludens-2 (ZO-2), GIAP C-terminus interacting protein (GIPC), calcium/calmodulin-dependent serine protein kinase (CASK), glutamate receptor interacting protein 2, (GRIP2) and Interleukin 16 (IL-16). A different pattern was observed for WNVNS5 as it associate with a broader repertoire of putative host PDZ proteins.

  • 11.
    Mlera, Luwanika
    et al.
    Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institutes of Health, Hamilton MT, USA.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper. Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institutes of Health, Hamilton MT, USA.
    Offerdahl, Danielle K.
    Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institutes of Health, Hamilton MT, USA.
    Dahlstrom, Eric
    Genomics Unit, Research Technologies Branch, National Institutes of Health, Hamilton MT, USA.
    Porcella, Stephen F.
    Genomics Unit, Research Technologies Branch, National Institutes of Health, Hamilton MT, USA.
    Bloom, Marshall E.
    Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institutes of Health, Hamilton MT, USA.
    Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line2016Ingår i: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 8, nr 9, artikel-id 252Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893AC (29% of 5978 reads at 12 h post infection (hpi)) and 2284TA (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.

  • 12.
    Tran, Pham Tue Hung
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Asghar, Naveed
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Karlsson, Anders
    Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; Nanoxis Consulting AB, Gothenburg, Sweden.
    Karlsson, Roger
    Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; Nanoxis Consulting AB, Gothenburg, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Screening of host proteins interacting with Kunjin, Langat, Zikareplication complex2019Ingår i: 16th Smögen Summer Symposium on Virology, 2019Konferensbidrag (Refereegranskat)
    Abstract [en]

    During infection and eclipse time, Flaviviruses induce invagination of the endoplasmic reticulum (ER) membrane to form compartments, protecting their viral replication complex. The rearrangements of ER membrane require modifications in ER membrane lipid constituents or binding of proteins to bend the membrane. Indeed, it has been implicated that both KUNV and DENV NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is not well known whether host proteins can also participate in the formation and maintenance of these compartments.In this project, we aimed to identify host proteins interacting with Kunjin, Langat, Zika replication complex. These proteins may function for ER invagination during Flavivirus infection. We used human adenocarcinoma epithelial A549 cells as a cell model, mosquito-borne Zika, Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, the ER membranes from infected and non-infected cells were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes to identify host candidate proteins that can cause the RC formation. To narrows the list of true candidate proteins, we attempted to enrich the RC-containing fractions by doing co-immuno precipitation. We are doing TMT-MS to identify and quantify the host proteins from Co-IP elutions. The functions of these proteins will be characterized by using molecular techniques.

  • 13.
    Tran, Pham Tue Hung
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Asghar, Naveed
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Karlsson, Roger
    Department ofClinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; NanoxisConsulting AB, Gothenburg, Sweden.
    Karlsson, Anders
    Department ofClinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; NanoxisConsulting AB, Gothenburg, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Identification and characterization of host proteins inducing the endoplasmic reticulum invagination during Flavivirus infection2019Ingår i: Positive-Strand RNA Viuses, 2019, s. 280-280Konferensbidrag (Refereegranskat)
    Abstract [en]

    When Flaviviruses infect host cells, they can induce invagination of endoplasmic reticulum (ER) membrane to form vesicle-like compartments. These unique structures are hypothetical to facilitate the viral replication by reducing diffusion of virus replication machinery and viral RNA, providing a scaffold to anchor the replication complex, and protecting viral RNA from host cell intrinsic surveillance. 

    The rearrangements of ER membrane to form these replication compartments (RCs) require modifications in its lipid constituents or binding of proteins to the membrane. Flaviviruses, indeed, use their proteins to generate RCs. It has been implicated that both KUNV and DENV viral NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is recondite whether host proteins can also participate in the formation and maintenance of RCs.

    In this project, we aimed to identify and characterize of host proteins inducing RC generation during Flavivirus infections. We used A549 as a cell model, and mosquito-borne Zika and Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, ER membranes were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes of infected cells and non-infected cells to identify host candidate proteins that can cause the RC formation.  We are attempting to enrich the RC-containing fractions and identifying proteins here, which narrows the list of true candidate proteins. The candidate proteins then will be characterized by using molecular techniques such as gene knock down, overexpression, and microscopy techniques.

  • 14.
    Tran, Pham Tue Hung
    et al.
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Asghar, Naveed
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Karlsson, Roger
    Department ofClinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; NanoxisConsulting AB, Gothenburg, Sweden.
    Karlsson, Anders
    Department ofClinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; NanoxisConsulting AB, Gothenburg, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Melik, Wessam
    Örebro universitet, Institutionen för medicinska vetenskaper.
    Screening of host proteins interacting with Kunjin, Langat, Zika replication complex2019Ingår i: Positive-Strand Rna Viuses, 2019Konferensbidrag (Refereegranskat)
    Abstract [en]

    When Flaviviruses infect host cells, they can induce invagination of endoplasmic reticulum (ER) membrane to form vesicle-like compartments. These unique structures are hypothetical to facilitate the viral replication by reducing diffusion of virus replication machinery and viral RNA, providing a scaffold to anchor the replication complex, and protecting viral RNA from host cell intrinsic surveillance. 

    The rearrangements of ER membrane to form these replication compartments (RCs) require modifications in its lipid constituents or binding of proteins to the membrane. Flaviviruses, indeed, use their proteins to generate RCs. It has been implicated that both KUNV and DENV viral NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is recondite whether host proteins can also participate in the formation and maintenance of RCs.

    In this project, we aimed to identify and characterize of host proteins inducing RC generation during Flavivirus infections. We used A549 as a cell model, and mosquito-borne Zika and Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, ER membranes were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes of infected cells and non-infected cells to identify host candidate proteins that can cause the RC formation.  We are attempting to enrich the RC-containing fractions and identifying proteins here, which narrows the list of true candidate proteins. The candidate proteins then will be characterized by using molecular techniques such as gene knock down, overexpression, and microscopy techniques.

  • 15.
    Wigerius, Michael
    et al.
    Södertörn University, Huddinge, Sweden; Dalhousie University, Halifax, Canada.
    Asghar, Naveed
    Södertörn University, Huddinge, Sweden.
    Melik, Wessam
    Södertörn University, Huddinge, Sweden.
    Johansson, Magnus
    Örebro universitet, Institutionen för hälsovetenskap och medicin.
    Scribble controls NGF-mediated neurite outgrowth in PC12 cells2013Ingår i: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 92, nr 6-7, s. 213-221Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neurite outgrowth is mediated by dynamic changes of the cytoskeleton and is largely controlled by Rho GTPases and their regulators. Here, we show that the polarity protein Scribble controls PC12 cell neurite outgrowth in response to nerve growth factor. Scribble knockdown decreases neurite numbers and increases neurite length. This effect is linked to TrkA the cognate receptor for NGF as pharmacological inhibition of phosphorylated TrkA (pTrkA) reduces Scribble expression. Moreover, Scribble forms a complex with the MAPK components ERK1/2 in a growth factor dependent manner. In RNAi experiments where Scribble expression is efficiently depleted sustained ERK1/2 phosphorylation is reduced. Conversely, siRNA with intermediate Scribble silencing efficiency fails to match this effect indicating that ERK1/2 activation depends on basic Scribble protein levels. Finally, Scribble translocates to the plasma membrane in response to growth factor where it complexes with HRas and Rac1 suggesting that the phenotype activated by loss of Scribble may be a result of altered GTPase activity. Together, these results demonstrate a novel role for Scribble in neurite outgrowth of PC12 cells. (c) 2013 Elsevier GmbH. All rights reserved.

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