As illustrated in the workflow sheet above, this project pursues different but interrelated goals. Chronologically, we will select a number of goats and sheep with serological profiles of difficult interpretation. These animals cause the most important problems to the Swiss SRLV reference center and are a source of great frustration for farmers and veterinary authorities.
1) We will systematically attempt to isolate the viruses infecting these animals.
a) Failure to isolate viruses from these animals, using the highly sensitive, broadly reacting RT activity test (PERT) described in the previous section, will provide strong evidence that some of the serological results (Idexx ELISA, WB and SU5 ELISA) may be false positive. We will explore the hypothesis that these particular SRLV strains are readily isolate from sheep but difficult to isolate from goats. Alternatively, these animals may have successfully controlled the primary infection, eliminating the infecting virus or restricting its replication below the limit of detection {Morin, 2003 9195 /id}.
b) The successfully isolated viruses will be characterized further.
2) Genomic fragments with diagnostic relevance, such as the SU5 region of env and particular regions of the gag and pol genes will be amplified by PCR, cloned and sequenced. The large abundance of starting material granted by the in vitro expansion of the infecting viruses in macrophage cultures will facilitate the PCR amplification using a panel of degenerated primers.
3) These sequences will be analyzed with different goals in mind:
a) A phylogenetic analysis will reveal the distance from and the classification of these sequences compared to known Swiss and international SRLV sequences.
b) Based on these sequences novel, genotype specific or broadly reacting serological diagnostic tools will be developed. This will permit a precise dissection of the epidemiological situation in particular flocks or geographic regions.
