Chikungunya is a mosquito-transmitted disease caused by Chikungunya virus (CHIKV). Symptoms of Chikungunya include debilitating joint pain and swelling, fever and rash. CHIKV was first discovered in 1953 in Tanzania, and has since caused periodic outbreaks of disease. The virus reemerged recently in 2004 and has since spread around the world affecting more than 3 million people. The different strains of CHIKV have been grouped into three phylogenetic clades: West African, Asian and East/Central/South African (ECSA). There are no FDA approved medicines or vaccines used to treat or prevent CHIKV infection. The antiviral drug, T-705 (commercially known as Favipiravir), has recently been shown to have activity against CHIKV. T-705 has already been approved in Japan for the treatment of influenza and is currently going through clinical trials in the US. Since there may be phenotypic differences between the clades of CHIKV, it is important to first characterize distinctions between the strains and determine the susceptibility of these strains to treatment. To do this, we obtained two different CHIKV strains from each of the three phylogenetic groups. These CHIKV strains displayed differences in their ability to replicate in cell culture and exhibited only slight differences in susceptibility to T-705 treatment. However, more profound differences were observed in mouse models where differences in disease severity and response to T-705 treatment were observed.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv., 2007.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniviversitet, 2006. / Härtill 7 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.

Master of Science / Grain Science and Industry / Cassandra K. Jones / Feed mill biosecurity is a growing concern for the feed industries, especially since the entry of Porcine Epidemic Diarrhea Virus (PEDV) to the United States. Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. However, research has confirmed swine feed and ingredients as potential vectors of transmission, so strategies are needed to mitigate PEDV in feed. The objective of the first experiment was to evaluate the effectiveness of various chemical additives to prevent or mitigate PEDV in swine feed and ingredients that had been contaminated post-processing. Time, formaldehyde, medium chain fatty acids, essential oils, and organic acids all enhance the degradation of PEDV RNA in swine feed and ingredients, but their effectiveness varies within matrix. Notably, the medium chain fatty acids were equally as successful at mitigating PEDV as a commercially-available formaldehyde product. Salmonella is also another potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella in ingredients and feed manufacturing is effective, but it does not eliminate the potential for cross contamination. Therefore, the objective of the second experiment was to evaluate the effectiveness of chemicals to mitigate Salmonella cross-contamination in rendered proteins over time. Both chemical treatment and time reduced Salmonella concentrations, but their effectiveness was again matrix dependent. Chemical treatment with medium chain fatty acids or a commercial formaldehyde product was most effective at mitigating Salmonella in rendered protein meals. The final experiment was conducted to evaluate the effectiveness of a dry granular acid, sodium bisulfate (SBS; Jones-Hamilton, Co., Waldridge, OH), to mitigate contamination of Salmonella in poultry feed. A surrogate organism, Enterococcus faecium, was utilized for this research in order to evaluate the effectiveness of SBS. Thermal processing, SBS concentration, and time all impacted biological pathogen levels in poultry diets, and including a dry granular acid may be an effective method to reduce pathogen risk. However, the most significant reduction of Enterococcus faecium was due to thermal mitigation. Notably, pelleting reduced Enterococcus faecium by 2-3 logs on day 0. In summary, both thermal processing and chemical inclusion can be used to reduce the risk of microbial pathogens in feed.