Influenza B virus contributes to a significant portion of influenza disease burden in men. It is structurally similar and replicates in the same manner as the influenza A virus, leading to a comparable clinical presentation between the two viral species. Since 1977, influenza B has caused seasonal epidemics around the world together with A/H1N1 and A/H3N2 subtypes, and has a strong affinity to affect children of school age and young adults. In the 1980s, two antigenically distinct lineages of influenza B virus emerged, one being the B/Yamagata lineage and the other known as B/Victoria lineage. The most significant antigenic difference between the two is located in the HA1 domain of the viral hemagglutinin. Host immunity is not shared between the two viral lineages. Therefore, the global prevalence of the two influenza B lineages is closely monitored by the World Health Organization in order to decide which viral lineage to include in the annual trivalent influenza vaccines. Surprisingly, the current methods used in influenza B viral surveillance and lineage discrimination have not seen much technical advancement in nearly 25 years since the emergence of two viral lineages. The current study presents a novel, asymmetric real-time PCR assay which is able to determine the viral lineage in addition to detecting the presence of influenza B virus in clinical specimens. Asymmetric PCR is performed by deliberately limiting the amount of primers in one side of a PCR reaction. This significantly affects the replication efficiency and sensitivity of the PCR reaction, but at the same time facilitates target sequence detection by hybridization probes, due to an increased number of single stranded products in the reaction. Nevertheless, the use of asymmetric PCR has been avoided in the past. The recent introduction of linear-after-the-exponential (LATE) PCR refines the method by adjusting melting temperature of PCR primers so that TmLimiting – TmExcess ≥ 0°C. The modification is shown to raise the efficiency of asymmetric PCR to those of symmetric PCR, as well as allowing more relaxed criteria for PCR primer and probe design. In the current asymmetric assay, pan-influenza B primers and probes targeting Victoria and Yamagata linage specific regions of the influenza B HA were evaluated against a similar symmetric influenza B assay published by the World Health Organization. HA plasmid standards and 155 clinical specimens were tested by both assays, in which the two had intra-assay CV% of less than 5%. Albeit the efficiency and sensitivity of WHO published assay was slightly higher, LATE-PCR based assay performed influenza B detection and genotyping simultaneously with the use of hydrolysis probes. The overall sensitivity/ specificity of the genotyping assay are 96.81%/100% while the WHO recommended assay is at 98.94%/100% for influenza B detection. The LATE-PCR based genotyping assay also successfully genotyped 89 out of 94 clinical specimens. In conclusion, the influenza B genotyping assay evaluated in this study performed favorably and could serve as an alternative to cumbersome viral culture methods to aid in high-throughput global influenza surveillance. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Bibliography: leaves 127-138. This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV.

Bibliography: leaves 127-138. / v, 138 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Bibliography: leaves 127-138. / v, 138 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Vaccinia virus (VV) is a large double-stranded DNA virus that is a prototypic member of the orthopoxvirus family. Previous works has showed that three of the major structural proteins found within the mature VV virion core 4a, 4b, and 25K are produced from higher molecular weight precursors at late times during infection and processed via a common morphogenic cleavage pathway that is intimately linked with virion assembly and maturation. The enzyme that carries out these cleavage reactions is unknown. A transient expression assay was used to demonstrate that the 17L gene product and its encoded cysteine proteinase activity is responsible for cleavage of each of the three major core protein precursors. Cleavage was demonstrated to occur at the authentic Ala-Gly-Xaa cleavage sites and require active enzyme. A truncated 17L protein lost the ability to cleave the core protein precursors. A conditional-lethal recombinant virus was constructed in which the expression of the 17L gene is under the control of the tetracycline operator/repressor system. In the absence of 17L expression, processing of the major VV core proteins is inhibited and electron microscopy revealed defects in virion morphogenesis prior to complete core condensation. Plasmid-borne 17L is capable of rescuing the growth of this virus. A structural model of 17L was developed and a unique chemical library was assayed for both cell toxicity and the ability to inhibit the growth of VV in tissue culture cells. A novel class of inhibitors was discovered that is capable of inhibiting VV. An in-vitro cleavage assay was developed to further characterize the activity of 17L. This assay is based on producing the major core protein precursors in a coupled transcription and translation assay and then mixing them with 17L enzyme extracts. Using this assay, 17L is shown to be capable of cleavage of each substrate. 17L is further characterized as a cysteine proteinase due to the inhibitory effects of known cysteine proteinase inhibitors such as NEM and iodoacetic acid, as well as through the use of specific small molecule inhibitors in this in-vitro assay. / Graduation date: 2005

The Feldmannia sp. virus is a large icosahedral virus that persistently infects marine brown alga Feldmannia sp.. So far, there is no information available about viral genome replication, gene structure and gene expression in this unique viral-host system. The purpose of this study was to characterize the general features of viral transcripts in the virus producing sporophyte plants. Northern analysis, using four cosmid clones that cover the entire viral genome, showed that there were six major transcripts and at least eighteen minor transcripts in the virus producing sporophyte plants. These transcripts are not evenly distributed in the viral genome. A 5.7 kb BamHI fragment-R was found to encode a 1.5 kb and a 0.9 kb major transcript, and those two major transcripts were chosen for detailed sequence analysis. The 1.5 kb transcript was identified as the putative major capsid protein (MCP) gene. The FsV MCP has significant similarity with the major capsid protein of Chlorella virus-PBCV-1 and with iridoviruses, fish lymphocystis disease virus, frog virus 3, and with African swine fever virus. / Graduation date: 1996

Copy of author's previously published work, inserted. / Bibliography: leaves 83-99. / ix, 99, [43] leaves, [29] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates an improved method with high efficiency for reassembly of CMV (Cucumber mosaic virus) and TAV (Tomato aspermy virus). Physico-chemical, serological and biological analyses show that the reassembled particles are indistinguishable from the native viruses. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1992

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Includes Appendix listing other publications by the author. Includes bibliographical references (leaves 134-181). Alfalfa mosaic virus was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms. Experiments showed that each isolate was biologically distinct and that the host range and symptomatology of each isolate was affected by the environmental condition.

Includes appendix containing journal publications co-authored by the author. Includes bibliographical references (leaves 130-151). Eighteen strains of Cucumber mosaic virus, including forteen from Australia, two from the USA, and two from Japan were used in this study.