Molecular characteristics of SRLV circulating in the post-eradication phase in Switzerland
To characterize the molecular features of the abovementioned attenuated SRLV strains, we fully sequenced 3 previously described SRLV A4 isolates. At first, the sequence analysis was unspectacular, showing that the three sequences were highly similar to each other and closely related to a previously reported SRLV A4 sequence. A more detailed analysis of their genomes revealed that the one isolate was most probably the product of a recombination event between the two other viruses, confirming the plasticity of these viruses. We then analyzed the virus sequences for the presence of intact ORF, looking in particular for genes encoding well known virulence factors, which were all present and intact. By contrast, the LTR region of these viruses presented several mutations potentially linked to the attenuated phenotype in vivo.
To test this hypothesis, we constructed an infectious molecular clone of the g6221 SRLV A4 isolate. This molecular clone replicates in fibroblastic cells and macrophages similarly to the wildtype virus. A detailed analysis of the promoter activity of the LTR revealed an AP-1 binding site that is crucial for the promoter activity in reporter gene assays as well as in the context of a replicating molecular clone. Other sites described in the literature appeared to be less crucial. Analysis of a unique AP-4 site showed a clear discrepancy between results obtained with reporter gene assays and mutated viruses. Within the limits of this in vitro study, however, we did not find evidence pointing to the LTR as the genetic correlate of attenuation for these viruses. Therefore our quest to unveil the molecular correlates of attenuation in these prominent SRLV A4 strains is continuing.