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Melik, Wessam
Publications (10 of 15) Show all publications
Tran, P. T., Asghar, N., Karlsson, R., Karlsson, A., Johansson, M. & Melik, W. (2019). Identification and characterization of host proteins inducing the endoplasmic reticulum invagination during Flavivirus infection. In: Positive-Strand RNA Viuses: . Paper presented at Positive-Strand RNA Viuses, KILLARNEY, Co.Kerry, Ireland, June 9-13,2019 (pp. 280-280).
Open this publication in new window or tab >>Identification and characterization of host proteins inducing the endoplasmic reticulum invagination during Flavivirus infection
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2019 (English)In: Positive-Strand RNA Viuses, 2019, p. 280-280Conference paper, Poster (with or without abstract) (Refereed)
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.

Keywords
Replication complex, Kunjin, Langat, Zika
National Category
Medical and Health Sciences Infectious Medicine
Research subject
Molecular Cellbiology; Biomedicine; Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-76406 (URN)
Conference
Positive-Strand RNA Viuses, KILLARNEY, Co.Kerry, Ireland, June 9-13,2019
Funder
Knowledge Foundation, HÖG15 20150201
Available from: 2019-09-13 Created: 2019-09-13 Last updated: 2019-09-19
Johansson, M., Frelin, L., Maravelia, P., Asghar, N., Melik, W., Caro-Perez, N., . . . Sallberg, M. (2019). Immunogenicity of a New Flaviviral-Based DNA Launched Suicidal Replicon for Protective Vaccination Against Hepatitis C. Paper presented at 22nd Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), Washington, DC, USA, April 29 - May 2, 2019,. Molecular Therapy, 27(4), 139-139
Open this publication in new window or tab >>Immunogenicity of a New Flaviviral-Based DNA Launched Suicidal Replicon for Protective Vaccination Against Hepatitis C
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2019 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 27, no 4, p. 139-139Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Cell Press, 2019
National Category
Medical Genetics Microbiology in the medical area
Identifiers
urn:nbn:se:oru:diva-73957 (URN)10.1016/j.ymthe.2019.04.004 (DOI)000464381001137 ()
Conference
22nd Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), Washington, DC, USA, April 29 - May 2, 2019,
Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-04-29Bibliographically approved
Asghar, N., Maravelia, P., Caro-Perez, N., Tarn, H., Melik, W., Pasetto, A., . . . Johansson, M. (2019). Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses. In: : . Paper presented at Positive-Strand RNA Viruses (E2), Killarney, Co. Kerry Ireland, June 9-13, 2019.
Open this publication in new window or tab >>Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses
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2019 (English)Conference paper, Poster (with or without abstract) (Refereed)
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.

National Category
Medical and Health Sciences Infectious Medicine
Research subject
Infectious Diseases; Immunology
Identifiers
urn:nbn:se:oru:diva-76208 (URN)
Conference
Positive-Strand RNA Viruses (E2), Killarney, Co. Kerry Ireland, June 9-13, 2019
Funder
Knowledge Foundation, HÖG15 20150201
Available from: 2019-09-10 Created: 2019-09-10 Last updated: 2019-09-11Bibliographically approved
Asghar, N., Maravelia, P., Caro-Perez, N., Tran, P. T., Melik, W., Pasetto, A., . . . Johansson, M. (2019). Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses. In: : . Paper presented at 16th Smögen Summer Symposium on Virology, Smögen, Sweden, August 22-24, 2019.
Open this publication in new window or tab >>Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses
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2019 (English)Conference paper, Poster (with or without abstract) (Refereed)
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.

National Category
Medical and Health Sciences Immunology Infectious Medicine
Research subject
Molecular Biology; Infectious Diseases; Immunology
Identifiers
urn:nbn:se:oru:diva-76634 (URN)
Conference
16th Smögen Summer Symposium on Virology, Smögen, Sweden, August 22-24, 2019
Available from: 2019-09-20 Created: 2019-09-20 Last updated: 2019-09-23Bibliographically approved
Tran, P. T., Asghar, N., Karlsson, R., Karlsson, A., Johansson, M. & Melik, W. (2019). Screening of host proteins interacting with Kunjin, Langat, Zika replication complex. In: Positive-Strand Rna Viuses: . Paper presented at Positive-Strand RNA Viuses, KILLARNEY, Co.Kerry, Ireland, June 9-13,2019.
Open this publication in new window or tab >>Screening of host proteins interacting with Kunjin, Langat, Zika replication complex
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2019 (English)In: Positive-Strand Rna Viuses, 2019Conference paper, Poster (with or without abstract) (Refereed)
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.

Keywords
Replication complex, Kunjin, Langat, Zika
National Category
Medical and Health Sciences Infectious Medicine
Research subject
Molecular Cellbiology; Biomedicine; Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-76400 (URN)
Conference
Positive-Strand RNA Viuses, KILLARNEY, Co.Kerry, Ireland, June 9-13,2019
Funder
Knowledge Foundation, HÖG15 20150201
Available from: 2019-09-13 Created: 2019-09-13 Last updated: 2019-09-13Bibliographically approved
Tran, P. T., Asghar, N., Karlsson, A., Karlsson, R., Johansson, M. & Melik, W. (2019). Screening of host proteins interacting with Kunjin, Langat, Zikareplication complex. In: 16th Smögen Summer Symposium on Virology: . Paper presented at 16th Smögen Summer Symposium on Virology, Smögen, Sweden, August 22-24, 2019.
Open this publication in new window or tab >>Screening of host proteins interacting with Kunjin, Langat, Zikareplication complex
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2019 (English)In: 16th Smögen Summer Symposium on Virology, 2019Conference paper, Oral presentation with published abstract (Refereed)
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.

Keywords
Replication complex, host cell, Westnile, Langat, Zika
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine; Molecular Cellbiology; Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-76402 (URN)
Conference
16th Smögen Summer Symposium on Virology, Smögen, Sweden, August 22-24, 2019
Funder
Knowledge Foundation, HÖG15 20150201
Available from: 2019-09-13 Created: 2019-09-13 Last updated: 2019-09-16Bibliographically approved
Asghar, N., Gunaltay, S., Tran, P. T., Melik, W., Höglund, U., Johansson, C., . . . Johansson, M. (2018). DNA launched suicidal flaviviruses as therapeutic vaccine candidates. In: : . Paper presented at 15th Smögen Summer Symposium on Virology, Smögen, Sweden, August 23-25, 2018.
Open this publication in new window or tab >>DNA launched suicidal flaviviruses as therapeutic vaccine candidates
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2018 (English)Conference paper, Poster (with or without abstract) (Refereed)
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.

National Category
Medical and Health Sciences Infectious Medicine Immunology
Research subject
Immunology; Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-76632 (URN)
Conference
15th Smögen Summer Symposium on Virology, Smögen, Sweden, August 23-25, 2018
Available from: 2019-09-20 Created: 2019-09-20 Last updated: 2019-09-23Bibliographically approved
Kellman, E. M., Offerdahl, D. K., Melik, W. & Bloom, M. E. (2018). Viral Determinants of Virulence in Tick-Borne Flaviviruses. Viruses, 10(6), Article ID 329.
Open this publication in new window or tab >>Viral Determinants of Virulence in Tick-Borne Flaviviruses
2018 (English)In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 10, no 6, article id 329Article, review/survey (Refereed) Published
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).

Place, publisher, year, edition, pages
MDPI, 2018
Keywords
tick-borne encephalitis virus, tick-borne flavivirus, virulence, neuropathogenesis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:oru:diva-68150 (URN)10.3390/v10060329 (DOI)000436146700052 ()29914165 (PubMedID)2-s2.0-85048705828 (Scopus ID)
Note

Funding Agency:

Intramural Research Program of NIH/NIAID

Available from: 2018-07-26 Created: 2018-07-26 Last updated: 2018-09-05Bibliographically approved
Mlera, L., Melik, W., Offerdahl, D. K., Dahlstrom, E., Porcella, S. F. & Bloom, M. E. (2016). Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line. Viruses, 8(9), Article ID 252.
Open this publication in new window or tab >>Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line
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2016 (English)In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 8, no 9, article id 252Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Basel: MDPI AG, 2016
Keywords
Langat virus genome, tick-borne flavivirus, persistent infection, Ixodes scapularis, ISE6 cells, deep-sequencing
National Category
Infectious Medicine
Identifiers
urn:nbn:se:oru:diva-53517 (URN)10.3390/v8090252 (DOI)000385396800012 ()2-s2.0-84987846223 (Scopus ID)
Note

Funding Agency:

Division of Intramural Research of the NIAID, NIH

Available from: 2016-11-15 Created: 2016-11-15 Last updated: 2018-07-17Bibliographically approved
Asghar, N., Lee, Y.-P., Nilsson, E., Lindqvist, R., Melik, W., Kröger, A., . . . Johansson, M. (2016). The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus. Scientific Reports, 6, Article ID 39265.
Open this publication in new window or tab >>The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 39265Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
London, United Kingdom: Nature Publishing Group, 2016
National Category
Microbiology
Identifiers
urn:nbn:se:oru:diva-54097 (URN)10.1038/srep39265 (DOI)000389971500001 ()27982069 (PubMedID)2-s2.0-85006377174 (Scopus ID)
Funder
Knowledge FoundationSwedish Research CouncilSwedish Foundation for Strategic Research
Note

Funding Agencies:

Foundation for Baltic and East European Studies

MIMS

Umeå Center for Microbial Research (UCMR)

Available from: 2016-12-21 Created: 2016-12-20 Last updated: 2018-07-23Bibliographically approved
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