Flock House virus (FHV) is a (+) ssRNA virus that belongs to the Nodaviridae family. The viral genome is composed of two viral RNA’s: RNA 1 and RNA 2. Deletion and mutation studies in the N- and C-terminus of protein alpha have identified protein regions required for the packaging of FHV viral RNAs. Residues 2-31 on the N-terminus have been attributed with RNA2 recognition, while residues at positions 32-50 were required for the packaging of RNA1. The C-terminus is needed for packaging of both viral RNAs. The identified protein regions involved in packaging viral RNAs bind random cellular RNA with high affinity and standard methods of identifying RNA-protein interactions such as gel shift mobility assays will be unable to discriminate between specific and unspecific binding. Due to the difficulty in differentiating between specific and unspecific binding a new method for studying RNA-protein interactions was developed using a surface based detection approach. The surface based system monitors real-time binding, whereby specific and unspecific RNA-protein interactions will be distinguished through comparison of relative association rates for each binding interaction. A well studied RNA-protein interaction, the HIV-1 Rev-RRE, was used to develop the methodology for the surface based system. Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein Rev that binds to HIV-1 mRNA of the Rev responsive element (RRE). Rev-RRE interaction regulates viral gene expression by controlling the export of spliced and unspliced mRNAs into the cytoplasm. The high-affinity and specificity of the Rev-RRE binding has been well characterized and was used as a model system to gauge the sensitivity of the surface based detection system, which can be further used to characterize various RNA-protein interactions. The surface based system uses diffractive optics to detect real time binding of molecules to receptors that are immobilized on a flat sensor surface. An avidin coated sensor surface was applied to couple the small biotinylated Rev peptide to the surface followed by binding its complementary RRE RNA. The binding interactions of the 30 nucleotide RRE to the immobilized 23 residue Rev peptide were successfully monitored using the avidin sensor. The Rev-RRE interaction was heavily influenced by the immobilization technique and steric hindrance at the sensor surface.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Biochemistry." Includes bibliographical references (139-146).

Title from title page of PDF (University of Missouri--St. Louis, viewed March 2, 2010). Includes bibliographical references (p. 58-62).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from OhioLINK abstract page. Abstract.

Thesis (M.S. in Computer Science)--Naval Postgraduate School, December 2003. / Thesis advisor(s): James B. Michael, Mikhail Auguston. Includes bibliographical references (p. 52-54). Also available online.

Thesis (M. Med. Sc.)--University of Hong Kong, 1983.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Human immunodeficiency virus type 1 (HIV-1) infection is a life-long threat which cannot be prevented by vaccination or completely cured by antiretroviral (ARV) treatment. The establishment of genotypic resistant testing (GRT) greatly improved the infection management and provided further guidelines on ARV strategies. The current study aimed to utilize the accumulated GRT data to investigate the HIV-1 molecular epidemiology, trends of drug resistance mutations (DRMs) among local patients and intra-host viral evolution. In order to maximize the therapeutic options for HIV-1 patients, the genotypic study also extended to two relatively new ARV classes for determination of integrase polymorphisms and tropism prevalence. From 1994 through 2013, a total of 3,108 plasma samples were available from 2,475 HIV-1 positive patients for epidemiological and viral investigations. The predominant genotypes in Hong Kong were subtype B (42.1%) and CRF01_AE (39.7%). Other genotypes including subtypes A1, C, D, F1, G, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC and CRF12_BF were also identified. Though the phylogenetic studies of subtype B and CRF01_AE identified several sporadic distinct clusters, their transmissions had been widely established between all local risk groups. In contrast, most of the non-B and non-AE cases were infected outside Hong Kong or circulated within non-Chinese population. However, one of the clusters in CRF07_BC suggested that this genotype might have been established quite well among local Chinese men-who-have-sex-with-men community. Routine GRT also provided valuable longitudinal data to study intra-host viral evolution. The observed intra-host evolutionary rates in CRF01_AE variants (11.68 x 〖10〗^(-4), IQR: 8.87 – 20.54 x 〖10〗^(-4) substitutions per site per year) was significantly higher than subtype B variants (5.27 x 〖10〗^(-4), IQR: 3.32 – 8.01 x 〖10〗^(-4) substitutions per site per year). Of 2,157 treatment-naïve patients included in this study, 4.4% of them were found carrying surveillance protease (PR) or reverse transcriptase (RT) DRMs. More importantly, half of the GRT data from 407 treatment-failure patients were potentially resistant to PR or RT inhibitors. The introduction of integrase inhibitors and CCR5 antagonists provided new insights and era for treating HIV-1 patients who were resistant to PR or RT inhibitors. The integrase GRT results showed that there were no major DRMs observed in integrase inhibitors-naïve patients. The distribution of integrase polymorphic sites between subtype B and CRF01_AE was significantly differed. On the other hand, the prevalence of X4/Dual-Mixed tropism in CRF01_AE variants was always significantly higher than subtype B, regardless of the interpretation algorithms and treatment history. In conclusion, this study demonstrated the HIV-1 molecular epidemiology and intra-host viral evolution of Hong Kong in the last two decades by utilizing the GRT data obtained from the ARV surveillance program. The in-house integrase GRT and genotypic tropism test were as well evaluated. The high prevalence of X4/Dual-Mixed tropism in CRF01_AE isolates would definitely limit the salvage therapeutic options for CRF01_AE-infected patients. Summarizing these findings, it is possible to project that CRF01_AE virus has unique characteristics from subtype B and should be investigated further in terms of disease progression and replicative capacity. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy