No description available.

No description available.

No description available.

No description available.

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

No safe and effective vaccine exists against respiratory syncytial virus (RSV), the main viral cause of lower respiratory tract infections in young children. Proteosome-based adjuvants, derived from the outer membrane proteins (OMP) of Neisseria species are potent inducers of mucosal and systemic immunity in humans and animals. RSV subunit vaccines based on enriched RSV proteins (eRSV) were formulated with proteosomes (Pro) or its S. flexneri LPS-supplemented derivative, Protollin (Prl). Administered intranasally (IN) in BALB/c mice, the vaccines elicited systemic and mucosal RSV-specific antibodies and fully protected against RSV challenge without enhanced pulmonary pathology or evidence of a Th2-biased response (eg: eosinophil infiltation or antigen-specific 1L-5 production by restimulated splenocytes or lung cells). Restimulation of cells from Prl-eRSV immunized mice elicited F peptide-specific CD8+ T cells producing IFNgamma and supernatant IFNgamma, TNFalpha, 1L-2 and IL-10. The Prl-eRSV vaccine was also studied in C57Bl/6 mice, to exploit the TLR2, TLR4 and MyD88-deficient (-/-) animals available on this background. Protection was significantly impaired in both TLR4-/- and MyD88 -/- mice, but not in TLR2 -/- mice following Prl- eRSV immunization and challenge. These studies revealed a role for the LPS component of Protollin in both initial (innate) cytokine release as well as dendritic cell maturation and Th1 polarization. Although antibody levels were sustained in MyD88-/- mice, the IgG1/IgG2a ratio was markedly higher in the absence of this pathway. The MyD88-/- mice also displayed elevated levels of pulmonary eosinophils following challenge, with concomitant reduction of neutrophils compared to wt mice. Using CD1d-iNKT cell-deficient mice (CD1-/-) in our BALB/c model, we also identified a significant role for the lipid component of both the Pro- and Prl-based vaccines. Responses to both vaccines in CD1-/- animals elicited lower antibody titers and reduced restimulated splenocyte supernatant cytokines (IFNgamma, IL-17 and IL-10), with concomitant augmentation of neutrophil recruitment (Prl only). Pro- and Prl-eRSV vaccines may therefore exert their powerful adjuvant effects by exploiting both CD1d-iNKT and, in the case of the Prlbased formulations the TLR4-MyD88-dependant signalling pathway. These pathways not only promote stronger Th1 immune responses but also act to control pulmonary eosinophil (MyD88-dependent) and neutrophil (MyD88 and CD1d-NKT-dependent) recruitment in a murine RSV challenge model.

Beak and feather disease virus (BFDV), a non-enveloped, icosahedral virus with a circular single stranded DNA (ssDNA) genome, is the causative agent behind psittacine beak and feather disease (PBFD), an often fatal disease affecting parrots. Symptoms include feathering abnormalities, loss of feathers, and occasionally beak and claw deformities. BFDV-induced immunosuppression results in an increased susceptibility to secondary microbial infections, which is often the cause of death in infected parrots. There is no cure, no effective treatment, and no protective vaccine for BFDV. The international trade in exotic parrots has facilitated the spread of BFDV, so that it now has a global presence. Given that over a quarter of the currently recognised 356 psittacine species are considered to be at risk of extinction in the wild, the worldwide presence of BFDV, coupled with its extreme environmental stability, poses serious concerns for the future of some of the worlds most endangered parrots. That genetic diversity exists among BFDV isolates has been established, yet in the 14 years since the genome was fully sequenced, very few full-length BFDV genome sequences have been deposited in GenBank, despite the technology to rapidly isolate and amplify entire circular ssDNA genomes being readily available. Most studies have sequenced just a portion of the genome, usually one of the open reading frames (ORFs) encoding the major viral proteins, to investigate phylogenetic relationships between isolates. However the two major BFDV ORFs, encoding the replication associated protein (Rep) and the capsid protein (CP), have been shown to evolve at different rates, with the functional Rep being generally more conserved while CP is more variable. When also considering the fact that ssDNA viruses are notoriously recombinant, it becomes clear that an analysis based on a portion of the genome is unlikely to accurately establish evolutionary relationships. Therefore the focus of the studies described in this thesis was on isolation and amplification of full-length BFDV genomes from avian blood and feather samples that first tested positive to a PCR-based BFDV screening method. Samples were collected by appropriately trained people in New Zealand, New Caledonia, and Poland, before being sent to the University of Canterbury for molecular and bioinformatic analysis. The sequences of the BFDV genomes from each region were compared to each other and to all other full BFDV genome sequences publically available in GenBank, to compare the genetic diversity among these isolates. Recombination analyses were also performed, to assess how recombination is impacting on the evolution of BFDV. New strains of BFDV and new subtypes of existing BFDV strains were discovered, indicating that the global genetic diversity may be greater than previously thought. Many strains also proved to be recombinants, in particular those from Poland. Europe has had a long history with importing and breeding exotic parrots, and the high degree of recombination among the Polish BFDV isolates coupled with the number of previously unsampled strains is an example of how maintaining populations of multiple species in captivity enables evolution through recombination, and emergence of novel viral strains. Full genome analyses can also enable tracking the source of an infection. A total of 78 full genome sequences from 487 samples tested were deposited into GenBank as a direct result of the work undertaken as part of this thesis, thereby adding to the existing knowledge base regarding BFDV. With continued global sampling and full genome analysis it may one day be possible to trace the history of BFDV to its original emergence.

No description available.

No description available.

INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use. METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured. RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied. CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.