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Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection
Thesis (PhD)--University of Pretoria, 2019.
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University of Pretoria
2019
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| _version_ | 1867613590676045824 |
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| access_status_str | Open Access |
| author2 | Paweska, Janusz Tadeusz |
| author_browse | Paweska, Janusz Tadeusz |
| author_facet | Paweska, Janusz Tadeusz |
| collection | Thesis |
| dc_rights_str_mv | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD)--University of Pretoria, 2019. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/70428 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:38:34.370Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/70428 Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection Paweska, Janusz Tadeusz monteirog2002@gmail.com Jansen van Vuren, Petrus Kortekaas, Jeroen Monteiro, Gaby Ermelindo Roberto UCTD Thesis (PhD)--University of Pretoria, 2019. Rift Valley fever virus (RVFV) is a mosquito-borne virus of the Phlebovirus genus, family Phenuiviridae, Order Bunyavirales. It has a negative-sense, single-stranded RNA genome, arranged in three segments; large (L), medium (M), and small (S). The S segment employs an ambisense coding strategy, coding for the nucleocapsid (N) protein in the negative sense orientation and the non-structural protein (NSs) in the positive sense orientation. The NSs protein is the major virulence factor of RVFV that inhibits host innate antiviral defense mechanisms. NSs suppresses the induction of type-I interferon (IFN) responses by forming a repressor complex on the IFN- promoter; promotes the degradation of specific proteins such as Protein Kinase R (PKR) and also induces a general transcription shut-off in infected cells. NSs contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Deletion of the NSs gene results in attenuation of RVFV, but less is known about the exact role of specific amino acid residues of the NSs protein. This thesis reports on clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild type RVFV (wtRVFV) and genetic mutants having cysteine-to-serine substitution at residues 39 and 40, 149, 178 and 194 of the NSs protein. The wtRVFV and four mutant viruses were rescued from transcription plasmids using a three plasmid-based reverse genetics system. BSR-T7/5 cells were infected with a recombinant fowlpox virus expressing T7 polymerase and subsequently transfected with pUC57 plasmids encoding RVFV S, M and L segments. The rescued viruses were used for animal experiments. BALB/c mice were inoculated subcutaneously (s.c.) with wtRVFV isolate 35/74 or one of the four mutants, and monitored for clinical signs, weighed and temperatures recorded. Samples were collected from mice at regular intervals post inoculation and from any moribund animals, including whole blood, liver, spleen, brain, lungs, heart and kidney tissues, for virus titration, histopathology and gene expression studies. Mice infected with the wtRVFV developed a fatal acute disease characterized by high levels of viral replication, severe hepatocellular necrosis, and up-regulation of transcription of genes encoding type-I and -II interferon (IFN), as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms underlying this attenuation were further investigated, including the ability of the mutant virus to suppress the type-I IFN response, trafficking to the nucleus, degradation of PKR and the functional transcription factor II Human (TFIIH) subunit p62. Expression levels of IFN-β mRNA transcripts were quantified in MRC-5 cells infected with the wtRVFV and the mutant RVFV-C39S/C40S. The results showed significant (p<0.05) upregulation of IFN- mRNA gene expression in the lysates of cells infected with the mutant C39S/C40S, while IFN- mRNA levels were reduced in wtRVFV infected cells. This result suggests that the C39S/C40S mutations compromise the antagonism of the IFN response by NSs. In vitro experiments in Vero cells demonstrated that both wtRVFV and the mutant C39S/C40S NSs proteins traffic to the nucleus and form filaments, although the mutant appeared less efficient in forming these structures. Infection of Vero cells with wtRVFV resulted in the degradation of PKR between 8 and 12 hours post infection hours post infection (hpi), whereas PKR levels were maintained in cells infected with mutant C39S/C40S. The attenuated phenotype of the mutant C39S/C40S virus is correlated with the compromised ability of mutant NSs to degrade PKR and to downregulate IFN- mRNA transcription. Veterinary Tropical Diseases PhD Unrestricted 2019-07-08T09:46:23Z 2019-07-08T09:46:23Z 2019/04/04 2019 Thesis Monteiro, GER 2019, Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/70428> A2019 http://hdl.handle.net/2263/70428 en © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title | Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title_full | Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title_fullStr | Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title_full_unstemmed | Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title_short | Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection |
| title_sort | mutation of adjacent cysteine residues in the nss protein of rift valley fever virus results in loss of virulence in a murine model infection |
| topic | UCTD |
| url | http://hdl.handle.net/2263/70428 |