Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease in the world and therefore is considered an emerging global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that infect humans are distinguished from one another by unique antigenic determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab) and cell mediated immune response in DENV infected hosts. The DENV E protein also mediates attachment of virions to host cell receptors and entry of virions into host cells by membrane fusion. The study of epitopes on DENV E protein is necessary for understanding viral function and for the design of unique polyvalent vaccines capable of inducing a neutralising antibody response against each DENV serotype. Reverse genetics using infectious cDNA clones has enabled the construction of functional intertypic DENV, where the E protein of one DENV serotype is put in the genetic background of a different DENV serotype. In addition, observations from our laboratory indicate that chimeric E proteins, consisting of E protein structural domains from different DENV serotypes can fold into functional proteins. This suggests that there is potential to engineer viruses with intertypic DENV E proteins as potential DENV vaccine candidates, which is the long term goal of studies within our research group. However, if a chimeric E protein was to be constructed containing epitopes involved in antibody mediated neutralisation of each DENV serotype, then knowledge of the location of these epitopes on the E protein of each DENV serotype would be essential. Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes involved in antibody mediated neutralisation on the E protein of all DENV serotypes, except DENV-4. The primary objective of this study was to identify epitopes on the DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and characterised using various serological and functional assays. The identification of DENV-4 specific neutralising MAbs in the panel was essential for subsequent experiments aimed at determining antigenic domains, structural domains or specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4. The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs were DENV-4 specific. All MAbs against the E protein recognised conformationally dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-adsorbent assay (ELISA). Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were DENV-4 specific and also demonstrated the strongest neutralisation activity of the panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of DENV-4. The MAbs that effectively captured DENV-4 were used in competitive binding assays (CBAs) to determine spatial relationships between epitopes and therefore define antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes recognised by the panel of MAbs segregated into two distinct domains (D4E1 and D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating human serum from DENV-4 infected patients suggested that the MAbs recognised the same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had experienced natural dengue infections, indicating the clinical relevance of such epitopes for the development of DENV vaccines. The reactivity of the capture MAbs with low pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8) recognised an acid resistant epitope. Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation followed the traditional epitope mapping approach of selecting subpopulations of DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a variety of strategies for mapping epitopes were used including DENV-4 variant analysis and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric DENV-3/4 E proteins and MAb screening of a bacterial peptide display library. DENV-4 variants including DENV-4 isolates from different geographical locations or chemically mutagenised DENV-4 were screened with neutralising MAbs to identify neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4 E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were essential for the binding of neutralising MAbs to an epitope. The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues within domains II and III of the DENV-4 E protein. No specific epitopes were identified for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E protein, when screened against DENV-3/4 chimeric DENV E proteins. The first epitope, which was recognised by the MAb F1G2, contained residue E95 which was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala) change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display library, which demonstrated binding of F1G2 to a peptide homologous with residues E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method. The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that F1G2 may be recognising a discontinuous epitope consisting of domains II and III. The second epitope, which was recognised by MAb 18F5, contained residue E329 which was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine (Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb 18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope. The potential mechanisms by which the DENV-4 specific MAbs neutralise virus infection were evaluated by the virus overlay protein binding assay (VOPBA). The binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the DENV-4 E protein did not prevent attachment of DENV-4 to the same protein. This was preliminary evidence that the binding of domain III epitopes by the MAbs F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb 13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are required for low pH mediated virus fusion to host cell membranes. Overall, the different methods used in this study identified epitopes involved in the neutralisation of DENV-4. The distribution of epitopes involved in neutralisation throughout the DENV-4 E protein were similar to the distribution of epitopes involved in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be possible to elicit neutralising antibodies against multiple DENV serotypes using chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate the design of novel tetravalent DENV vaccines.

abstract: Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

>Magister Scientiae - MSc / Acute respiratory infections (ARI) continue to be the leading cause of acute illnesses worldwide and remain the most important cause of infant and young children mortality. Many viruses such as rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, adenoviruses and coronaviruses are deemed to be the etiological agents responsible for ARI’s in children. The recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 contribute significantly to the hospitalization of children with ARI’s. HCoV-NL63 was first identified in 2004, as the pathogen responsible for the hospitalization of a 7 month old child presenting with coryza, conjunctivitis and fever. Since then a significant amount of knowledge has been gained in the clinical spectrum on this virus, however HCoV-NL63 is still not well characterized on the molecular and proteomic level. This dissertation focuses on bringing about this characterization by cloning the HCoV-NL63 Nucleocapsid gene to be expressed in a bacterial system and transfecting the Nucleocapsid, Membrane and Envelope genes into a Mammalian cell culture system in order for its respective proteins to be expressed. With the use of Bioinformatic analytic tools certain characteristics of HCoV-NL63 Nucleocapsid, Membrane and Envelope proteins are able to be identified, as well as certain motifs and/or regions that are important in the functioning of these proteins. By comparing the results obtained for HCoV-NL63 N,M and E to other well studied coronavirus homologous will enlighten us on the potential role(s) of these proteins in determining HCoV-NL63 pathogenicity and infectivity. vi Although certain functions of these proteins can be deduced by the means of bioinformatics analysis, it is still imperative for it to be extensively characterized In Vitro. This will therefore form a fundamental step in the development of many other projects, which unfortunately fall outside the scope of this M.Sc thesis.