Characterisation of equine infectious anaemia virus (EIAV) strains.

This project focused on two facets of equine infectious anaemia virus (EIAV) biology; genomic characterisation and virus isolation. A lack of published full genome sequences has led to a poor understanding of genomic variation in the field and inability to design molecular detection methods. Equally the technically demanding nature of equine macrophages has led to much research EIAV being conducted using cell lines that cause viral adaption. Post mortem tissues from six and serum from one British outbreak cases were used to both sequence the full genome of novel field strains and develop a reproducible cell culture system that minimises adaption. Sequencing using primer walking and Sanger sequencing yielded the gag and pol of a symptomatic case but the high variability of the env prevented effective primer design. Next generation sequencing (NGS) was then used to avoid the requirement for sequence specific primers. The full genomes of the three symptomatic viruses were resolved. Two asymptomatic cases were also sequenced but no virus specific reads were returned. One symptomatic virus yielded a high coverage allowing a population analysis which showed the majority of variants localised to the gp90 glycoprotein. Each sequenced British genome added a new EIAV phylogenetic group with each group showing nucleic acid divergence of ~30% from the others. To develop a culture system that minimises adaption, primary monocytes were differentiated to macrophages using M-CSF and autologous equine serum, and successfully infected with the Wyoming strain. The system was used to successfully isolate virus from horses with clinical signs of infection. The activation of the macrophages had little effect on virus replication and dendritic cells appeared to be unable to support efficient replication. Variation was seen between different monocyte isolations so the effect of single cytokines was tested, with IL-4 found to improve EIAV replication reproducibly.