Antigenic, genetic and biological characterization of the flavivirus Alfuy.

May, Fiona J. (2006). Antigenic, genetic and biological characterization of the flavivirus Alfuy. PhD Thesis, School of Molecular and Bicrobial Science, The University of Queensland.

       
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Author May, Fiona J.
Thesis Title Antigenic, genetic and biological characterization of the flavivirus Alfuy.
School, Centre or Institute School of Molecular and Bicrobial Science
Institution The University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Roy A. Hall
Total pages 220
Collection year 2006
Language eng
Subjects L
270206 Genetic Immunology
780105 Biological sciences
Abstract/Summary Alfuy virus (ALFV) is a member of the Japanese encephalitis virus (JEV) antigenic complex of flaviviruses and is currently classified as a subtype of Murray Valley encephalitis virus (MVEV). However, while MVEV and JEV pose significant health risks to regions of Northern Australia, there is little evidence that ALFV causes disease in humans or other animals. Due to the apparent low pathogenicity of ALFV, few studies have previously focused on this virus, therefore, very little is known about it. This thesis aims to improve our understanding of this virus, with the potential of using ALFV, or markers of attenuation discovered within its genome, to develop novel vaccines for use against MVEV, JEV, and even other members of the Flavivirus genus. Previously, nucleotide sequence analysis of a small portion of the genome of ALFV was used to reclassify the virus as a subtype of MVEV. During this project the entire genome of ALFV was sequenced and compared with other members of the genus. Phylogenetic relationships revealed that although ALFV is closely related to MVEV, it is no closer to MVEV than to JEV or to Usutu virus, suggesting that ALFV should not be classed as a subtype of MVEV. Indeed, antigenic studies comparing ALFV, MVEV and JEV, using mAbs produced to MVEV, show a distinct antigenic profile for ALFV. Several unique genetic markers that may affect the virulence of ALFV were observed in the genome. Within the envelope (E) protein, the conserved Nlinked glycosylation motif contained a substitution causing a loss of glycosylation. Also within the E protein, the hinge region, involved in low-pH dependent conformational changes in the protein, also showed several amino acid differences when compared with MVEV and other related viruses. In addition, the highly conserved terminal dinucleotide was deleted. This region is thought to be important for replication of viral RNA. Virulence studies of different strains of ALFV in three-week old Swiss outbred mice also show a different pattern of virulence to MVEV. While MVEV is highly neurovirulent and neuroinvasive in these animals, ALFV exhibits weak neuroinvasiveness, producing very little disease in mice infected intraperitoneally, and producing ALFV-specific antibody only in mice infected with high doses. Even in the highly susceptible IFN-alphaR-/- mice (deficient in the receptor for interferon-alpha), ALFV caused significantly delayed mortality. This data suggests that ALFV is inefficient at replicating in the periphery of these animals. ALFV also exhibited a different tropism to MVEV in vitro, showing significantly reduced replication in Vero cells, a line of African Green Monkey kidney cells that supports efficient replication of all other members of the JEV complex. Binding studies showed that ALFV was able to bind to the surface of these cells, but electroporation of viral RNA directly into the cytoplasm was unable to initiate infection, suggesting inefficient replication or translation of ALFV viral RNA in this cell type. To determine the motifs responsible for these unique phenotypic characteristics of ALFV, three chimeric viruses were engineered based on a previously constructed infectious clone of MVEV. One, containing the structural (prM and E) genes of ALFV in the MVEV backbone, exhibited weak neuroinvasiveness, similar to ALFV, but unlike ALFV, was able to replicate efficiently in Vero cells, and produced ALFVspecific antibody in mice infected with low viral doses. This suggests that the structural genes of ALFV are associated with poor neuroinvasion, but are not responsible for poor extraneural replication in mice, or lack of growth in Vero cells. Another mutant virus was constructed by deleting the terminal dinucleotide in the MVEV infectious clone. This clone spontaneously reverted to the wild type sequence, preventing phenotypic analysis of this mutation. The final chimera contained the nonstructural genes of ALFV with the structural genes and the UTRs of MVEV. Unfortunately, despite several attempts, this clone was unstable in the bacterial host, and insertions or deletions were spontaneously introduced into the genome. Based on phylogenetic, antigenic and virulence data, ALFV is sufficiently different from MVEV to be classified as a separate virus within the genus. Determinants of the low neuroinvasiveness of ALFV have been localized to the structural genes of ALFV, probably within the hinge region of E, and/or due to the lack of glycosylation of the E protein. The cause of the delayed growth in Vero cells was unable to be determined, but is not due to a motif within the structural genes.

 
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Created: Fri, 21 Nov 2008, 21:07:43 EST