Publications

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-gamma, responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-gamma ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-gamma response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells. A range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy. Significant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions. These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.

For the first time, complete genome sequences of four lineage III peste des petits ruminants (PPR) viruses (Oman 1983, United Arab Emirates 1986, Ethiopia 1994, and Uganda 2012) originated from the Middle East and East Africa are reported here. The availability of complete genome sequences from all four lineages (I to IV) of the PPR virus (PPRV) would greatly help in a comprehensive understanding of the molecular evolution and emergence of PPRV.

In the past decade biting midges of the subgenus Avaritia (Diptera: Ceratopogonidae) have been popular subjects of applied entomological studies in Europe owing to their implication as biological vectors in outbreaks of bluetongue and Schmallenberg viruses. This study uses a combination of cytochrome oxidase subunit I barcode sequencing and geometric morphometric analyses to investigate wing shape as a means to infer species identification within this subgenus. In addition the congruence of morphological data with different phylogenetic hypotheses is tested. Five different species of the subgenus Avaritia were considered in the study (C. obsoletus (Meigen); C. scoticus Kettle and Lawson; C. chiopterus (Meigen); C. dewulfi Goetghebuer and C. imicola (Kieffer)). The study demonstrated that over 90% of individuals could be separated correctly into species by their wing shape and that patterns of morphological differentiation derived from the geometric morphometric analyses were congruent with phylogenies generated from sequencing data. Morphological data produced are congruent with monophyly of the subgenus Avaritia and the exclusion of C. dewulfi from the group containing C. obsoletus, C. scoticus and C. chiopterus. The implications of these results and their importance in a wider context of integrating multiple data types to interpret both phylogeny and species characterization is discussed.