Discovery of a novel iflavirus sequence in the eastern paralysis tick Ixodes holocyclus

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus

Commensal viruses of mosquitoes: Host restriction, transmission, and interaction with arboviral pathogens

Recent advances in virus detection strategies and deep sequencing technologies have enabled the identification of a multitude of new viruses that persistently infect mosquitoes but do not infect vertebrates. These are usually referred to as insect-specific viruses (ISVs). These novel viruses have generated considerable interest in their modes of transmission, persistence in mosquito populations, the mechanisms that restrict their host range to mosquitoes, and their interactions with pathogens transmissible by the same mosquito. In this article, we discuss studies in our laboratory and others that demonstrate that many ISVs are efficiently transmitted directly from the female mosquito to their progeny via infected eggs, and, moreover, that persistent infection of mosquito cell cultures or whole mosquitoes with ISVs can restrict subsequent infection, replication, and transmission of some mosquito-borne viral pathogens. This suggests that some ISVs may act as natural regulators of arboviral transmission. We also discuss viral and host factors that may be responsible for their host restriction

Approaches for the development of rapid serological assays for surveillance and diagnosis of infections caused by zoonotic flaviviruses of the Japanese encephalitis virus serocomplex

Flaviviruses are responsible for a number of important mosquito-borne diseases of man and animals globally. The short vireamic period in infected hosts means that serological assays are often the diagnostic method of choice. This paper will focus on the traditional methods to diagnose flaviviral infections as well as describing the modern rapid platforms and approaches for diagnostic antigen preparation. Copyrigh

Characterisation and recombinant expression of antigens for the rapid diagnosis of West Nile virus infection

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

A whole-blood homogeneous assay for the multiplex detection of the factor V G1691A and the prothrombin G20210A mutations

We describe an assay for the simultaneous detection in DNA or whole blood samples of the G1691A (Leiden) mutation in the factor V allele and the G20210A mutation in the prothrombin allele, both of which predispose humans to thrombosis. The assay uses a dual-probe quenching system in which one probe of a pair is labelled with a fluorophore and the adjacent probe is labelled with a fluorescence quencher. The primer partners were present in unequal amounts, and the genotypes were identified by melt curve analysis, which, together with the PCR, was performed in a Rotor-Gene™ centrifugal thermal cycler. The performance of the assay was assessed by consistency of melting temperature (Tm) values and height of melt curves from within- and between-run analyses. The stability of the PCR reagents and whole blood was assessed by determining Tm and peak height values after storage at −20, 4 °C or room temperature. Concentrations of the reaction reagents were finely adjusted to obtain consistent and clearly distinguishable melt curves that facilitated automatic assignment of 43 genotypes comprising wild-type and mutant alleles. The mean (SD) Tms and peak heights of 31 DNA samples assayed in a single run were 54.85 °C (0.07) and 2.33 U (0.15) for factor V and 53.69 °C (0.19) and 1.50 U (0.08) for prothrombin. The Tms and melt peak heights for DNA from wild-type individuals (n=10) when assayed for between- and within-run variability showed SDs of ≤0.17 and ≤0.3 for Tm and peak height values, respectively. When whole blood was used as the sample, the assay performance was comparable to that of purified DNA samples, with whole blood assays (n=30) yielding mean (SD) Tms and peak heights of 53.74 °C (0.11) and 2.10 U (0.32) for factor V and 53.27 °C (0.12) and 2.15 U (0.18) for prothrombin. Reaction reagents stored for 12 months at room temperature produced satisfactory results, as did whole blood stored frozen for 2 years or in mineral oil at room temperature for at least 1 week. The features of the homogeneous PCR assay described in this article should facilitate the use of such assays in clinical laboratories