Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 116-119).

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Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, August 2009. / Major professor: Kurt E. Gustin. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

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A new aphid virus, aphid lethal paralysis virus (ALPV), was isolated from laboratory-propagated Rhopalosiphum padi aphids co-infected with R. padi virus (RhPV). ALPV and RhPV were separated and ALPV was characterised in detail. Virions are isometric with a diameter of 26 nm, a sedimentation coefficient of 164 Sand a density in CsCl of 1.34 g/ml. Virions contain a 9.7 kb polyadenylated, singlestranded RNA and three major proteins with molecular weights of approximately 30 kilodaltons. By characterising RhPV further, two additional putative capsid proteins were found, an RNA poly(A) tract was detected and an RNA size of 10 kb was determined. A South African isolate of RhPV (RhPVoFs) was found to be serologically identical but physically distinct from a USA isolate. Complementary DNA was synthesized from RhPVOFS RNA and cloned into the plasmid vector, pBR322. This clone was used for the detettion of virus in aphids. ALPV and RhPV are serologically unrelated. ALPV is serologically distantly related to two insect picornaviruses, cricket paralysis virus (CrPV) and Drosophila C virus. No nucleic acid homology was detected between ALPV cDNA and CrPV by dot-blot hybridization. ALPV is serologically unrelated to seven other insect picornalike viruses. RhPV is serologically unrelated to any of the above mentioned viruses. ALPV and RhPV RNAs were efficiently translated in rabbit reticulocyte lysate into high molecular weight polypeptides, the sum of which exceeded the coding capacity of the genomes. Putative capsid precursor proteins of ALPV and RhPV were identified by immunoprecipitation. ALPV translation products were post-translationally cleaved as demonstrated in pulse-chase experiments and in experiments using a translation inhibitor. The efficiency of cleavage was concentration-dependent indicating the action of a protease. In parallel experiments with RhPV RNA, no evidence of post-translational cleavage was observed. In a survey of aphids collected in South Africa, ALPV and RhPV were detected in aphids from two major small-grain producing areas. Both viruses were found to naturally infect most of the cereal aphid species found in this country. ALPV and RhPV infections of R. padi resulted in a marked reduction in longevity and fecundity relative to uninfected aphids. Both viruses were found to be horizontally and vertically transmitted through aphid populations, and aphid host plants and aphid predators could be implicated in virus dissemination. ALPV and RhPV have many properties in common with each other as well as with insect and mammalian picornaviruses. Based on this data, it is proposed that ALPV and RhPV be classified into the picornavirus group (family Picornaviridae).

According to the World Health Organization, over 200 infectious diseases in humans originate from animals (zoonoses), posing significant threats to human health. Zoonotic agents account for the majority of emerging and re-emerging pathogens. The human-animal interface has been recognised as an important risk factor that facilitates viruses to cross the species barrier and establish infection in humans. This indicates a need to perform surveillance of human populations who are at high risk of zoonotic infection due to their frequent contact with animals, together with the animals to which humans are exposed. The VIZIONS (Vietnam Initiative on Zoonotic Infections) has been conducted to directly respond to that need. The large virus family Picornaviridae include known emerging pathogens that have major impacts on the economies and human and animal health (e.g. foot-and-mouth disease virus, hand foot and mouth disease virus). Some enteroviruses (EVs) and parechoviruses in this family have been shown to be able to infect both humans and animals while a number of new picornaviruses (new EV variants, cosaviruses, cardioviruses, hunniviruses) with unknown pathogenicity and zoonotic potential have been discovered. This thesis, as part of VIZIONS, hopes to address the following gaps in our knowledge of such viruses in six genera (Enterovirus, Parechovirus, Cosavirus, Cardiovirus, Kobuvirus and Hunnivirus) of the family Picornaviridae: 1) The prevalence and genetic diversity of picornaviruses in studied samples 2) The epidemiology and disease association of the identified viruses 3) The overlaps (if any) of picornaviruses circulating in animals and humans 4) Possible animal sources of picornavirus infections in humans In order to do that, over 2,000 faecal samples collected from a wide range of hosts (pigs, rats, bamboo rats, shrews, bats, chickens, ducks, boars, civets, porcupines, monkeys and humans) were screened for picornaviruses by nested PCR and real-time PCR assays. Detection frequencies varied between viruses and sample origins with kobuvirus as the most commonly detected virus, followed by EV, cardiovirus and hunnivirus. Parechovirus and cosavirus were not detected. Comparison of detection frequencies of viruses infecting pigs revealed a disease (diarrhoea) association with porcine kobuvirus (PKV) but not EV infections. However, differences in PKV viral loads between diarrhoeic and non-diarrhoeic pigs were not statistically significant (p = 0.22). In addition, the PKV VP1 sequences from the two pig categories were not phylogenetically distinct. EV VP1 sequences obtained from pigs and boars showed high genetic diversity with four previously known types and nine new types (EV-G8 to -G16). Analyses of complete genome sequences of two new EV types provided evidence for inter-type recombination with a putative breakpoint in the 2A coding region. Similarly, study on samples from monkeys showed endemic infection of EV but no overlap with EV variants in humans was observed. The majority of EV detected in monkeys were novel with evidence for chimeric genomes and putative recombination breakpoints in the 2A region. New criteria for the classification of EV were additionally proposed. Characterization by sequencing of VP4/VP2 and VP1 regions or complete genomes of picornaviruses in rats and bamboo rats also showed relatively high genetic diversity. While these viruses can infect different species of rats, they were again genetically different from viruses detected in the studied human populations. In summary, studies in this thesis provide substantial new information on the prevalence, genetic diversity and disease association of picornaviruses in the studied populations. However, picornaviruses detected from animals were consistently separate from those found in humans, consistent with a relatively limited zoonotic potential of members of the virus family.

Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.