Some viruses which infect plants, animals and man are transmitted by an intermediary arthropod vector. The viruses for which this is true are termed arboviruses (Arthropod-borne-viruses). In many instances the virus delivered to the new host results in the establishment of a disease state and/or the death of the infected host. In all instances, however, the arthropod (invertebrate) vector is apparently unaffected by the virus it is carrying. One family of viruses which are transmitted to their vertebrate host via an arthropod vector is the virus family Bunyaviridae, in this dissertation specifically the viruses Inkoo and Uukuneimi are described. The characteristics of Inkoo and Uukuneimi growth in both vertebrate baby hamster kidney (BHK-21, WI2) and invertebrate Aedes albopictus (mosquito) cell cultures has been examined. Vertebrate cells supported, to a high titer, the growth of both Inkoo and Uukuneimi virus while Aedes albopictus cell cultures supported high titer growth of Inkoo but not Uukuneimi. In both cases, however, the vertebrate cells were killed as a cosequence of infection where as, the invertebrate infection did not result in cell death or in detectable cytopathic effect. The invertebrate cells infected with either Inkoo or Uukuneimi continue to grow and also continue to express virus specific (actinomycin D resistent) RNA synthesis. The virus infected invertebrate cells are characterized as being persistently infected because of their resistence to homologous virus superinfection and by detectable virus specific RNA synthesis. Virus released from the Inkoo persistently infected cells displays a heterogeneous plaque morphology as well as temperature sensitive virus plaque mutants. Virus particles released from the Inkoo persistently infected Aedes albopictus cells are considered defective interfering-like. The RNA profile both intracellularly and of released virus particles from the persistently infected cell cultures is different from that observed during vertebrate cell culture infections. Cell death resulted from infection of BHK-21 WI2 cells with virus from Inkoo persistently infected Aedes albopictus cell cultures. The virus plaque morphology and RNA profile is similar to standard virus infection of BHK-21 WI2 cells. The experiments with tissue culture virus-cell systems aids in understanding the natural transmission of arboviruses between the vertebrate-invertebrate portions of the arbovirus natural life-cycle.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 109-129).

Call number: LD2668 .T4 1979 T516 / Master of Science

The viral dynamics of the hepatitis C virus (HCV) in newly acquired infection are not well understood. HCV exists within an individual as a spectrum of minor variants termed quasispecies. The evolution of minor variants may contribute to viral escape of the host?s immune response, thereby facilitating development of chronic infection. The hypervariable 1 region (HVR1) is the most heterogeneous part of the HCV genome and contains a putative B-cell epitope. Thus, diversity in HVR1 could be a strategy used to evade neutralising antibodies. Acutely infected individuals (n=24) were examined with the aim of defining HVR1 quasispecies diversity in acute infection. The characterisation of the E1/HVR1 sequence and host specific evolution of HCV minor variants in treatment nonresponders was also investigated. HCV E1/HVR1 fragments were amplified from 48 sera using a combined reverse transcription-polymerase chain reaction (RT-PCR). Products were TA cloned into pCRIITOPO and approximately 10-20 clones were sequenced from each sample. HVR1 quasispecies diversity was examined longitudinally via sequence analysis. Quasispecies diversity was characterised primarily by mean nucleotide diversity. The mean HVR1 diversity of the acute cohort (n=48; 2.12% ?? 2.22) was lower than the diversity obtained for a cohort of chronically infected individuals (n=99; 4.5% ?? 5.1). There was no significant difference in mean HVR1 diversity between the HIV/HCV co-infected and HCV mono-infected groups (p=0.99) or between the clearer and non-clearer groups (p=0.85). Examination of amino acid usage and the hydropathic profile of each position in HVR1 revealed that sequence variation was confined to specific sites. The investigation of host specific evolution of HVR1 quasispecies demonstrated that minor variants (comprising 10- 20% of a population) became the dominant species over time in two treatment non-responders. These variants bore mutations that were not reflected in the consensus sequence of their respective populations at the initial timepoint analysed. Common infection was identified by 98% HVR1 sequence homology within two pairs of individuals. The evolution of common strains appeared to be different between individuals, suggesting host pressures may influence quasispecies evolution. This thesis provided an insight into the viral dynamics and host specific evolution of acute phase quasispecies.

Entry of herpesviruses is mediated by the interactions between viral glycoproteins and cellular receptors. Among these glycoproteins, gB plays an important role. In this study, my major focus was to study gB's functions in the virus entry process and the structural requirements for gB to conduct its functions. The virus model in my study is bovine herpesvirus 1 (BHV-1), a member of the alphaherpesviruses. BHV-1 gB is a type I integral membrane protein with a potential transmembrane anchor at the C-terminal region. A cleavage site in the middle divides this molecule into two subunits, gBb and gBc. In this study, a truncated gB, gBt (residues 1 to 763), and N-terminal subunit, gBb (residues 1 to 505), were first expressed under the control of the bovine heat-shock protein 70A (hsp70A) gene promoter in stably transfected Madin Darby bovine kidney (MDBK) cells. Both forms of gB were secreted into the medium with apparent molecular weights as anticipated, and they were reactive to all gB-specific monoclonal antibodies used in this study. Affinity-purified gBt and gBb were able to elicit antibody responses in mice to an extent comparable to those induced by authentic gB. These results suggest that gBt and gBb retain the structural and antigenic properties of authentic gB. Furthermore, the intracellular processing of gBt and gBb was similar to that of authentic gB in virus-infected cells. Finally, gBt was proteolytically cleaved after conversion of the high mannose-containing precursor to the mature form. These truncated gBs that were prepared served as reagents for the core of my studies. BHV-I gB can bind to heparin sulfate (HS) and another non-HS receptor on MDBK cells. We assume that high-affinity binding to the non-HS receptor is important for BHV-1 infectivity. BHV-1 gB forms dimers in infected cells and in virions, and its dimerization domain may be located between residues 506 to 763. The cytoplasmic domain of BHV-1 gB is important for the existence of the high-affinity binding site. Without the cytoplasmic domain, the truncated gB derivatives exhibit conformational changes and loss of the high-affinity binding site. By comparing the expression of different gB derivatives in MDBK cells, it was found that in the putative transmembrane region, segment 3 is the real membrane anchor, whereas segment 2 is the fusogenic domain. (Abstract shortened by UMI.)

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 174-194). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 174-194). Also available in print.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The viral dynamics of the hepatitis C virus (HCV) in newly acquired infection are not well understood. HCV exists within an individual as a spectrum of minor variants termed quasispecies. The evolution of minor variants may contribute to viral escape of the host?s immune response, thereby facilitating development of chronic infection. The hypervariable 1 region (HVR1) is the most heterogeneous part of the HCV genome and contains a putative B-cell epitope. Thus, diversity in HVR1 could be a strategy used to evade neutralising antibodies. Acutely infected individuals (n=24) were examined with the aim of defining HVR1 quasispecies diversity in acute infection. The characterisation of the E1/HVR1 sequence and host specific evolution of HCV minor variants in treatment nonresponders was also investigated. HCV E1/HVR1 fragments were amplified from 48 sera using a combined reverse transcription-polymerase chain reaction (RT-PCR). Products were TA cloned into pCRIITOPO and approximately 10-20 clones were sequenced from each sample. HVR1 quasispecies diversity was examined longitudinally via sequence analysis. Quasispecies diversity was characterised primarily by mean nucleotide diversity. The mean HVR1 diversity of the acute cohort (n=48; 2.12% ?? 2.22) was lower than the diversity obtained for a cohort of chronically infected individuals (n=99; 4.5% ?? 5.1). There was no significant difference in mean HVR1 diversity between the HIV/HCV co-infected and HCV mono-infected groups (p=0.99) or between the clearer and non-clearer groups (p=0.85). Examination of amino acid usage and the hydropathic profile of each position in HVR1 revealed that sequence variation was confined to specific sites. The investigation of host specific evolution of HVR1 quasispecies demonstrated that minor variants (comprising 10- 20% of a population) became the dominant species over time in two treatment non-responders. These variants bore mutations that were not reflected in the consensus sequence of their respective populations at the initial timepoint analysed. Common infection was identified by 98% HVR1 sequence homology within two pairs of individuals. The evolution of common strains appeared to be different between individuals, suggesting host pressures may influence quasispecies evolution. This thesis provided an insight into the viral dynamics and host specific evolution of acute phase quasispecies.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.