Genetic identification of two Acipenser iridovirus-European variants using high-resolution melting analysis

Acipenser iridovirus-European (AcIV-E) is an important pathogen of sturgeons. Two variants differing by single-nucleotide polymorphisms (SNP) in the Major Capsid Protein gene have been described, but without any indication as to their prevalence in farms. To facilitate epidemiological studies, we developed a high-resolution melting (HRM) assay to distinguish between two alleles (var1 and var2) differing by five point substitutions. The HRM assay detected as little as 100 copies of plasmids harboring cloned sequences of var1 and var2, which have melting temperatures (Tm) differing by only 1 °C. The assay was specific of AcIV-E as demonstrated by the absence of signal when testing a related, yet distinct, virus as well as DNA from an AcIV-E-negative sturgeon sample. Experiments with mixtures of two distinct plasmids revealed abnormal melting curve patterns, which showed dips just before the main melting peaks. These dips in the curves were interpreted as the dissociation of heteroduplexes fortuitously created during the PCR step. Screening AciV-E-positive field samples of Russian sturgeons from three farms revealed the presence of var2, based on the Tm. However, for a few samples, the melting curves showed patterns typical of var2 as the dominant viral genome, mixed with another minor variant which proved to be var1. In conclusion, HRM is a simple method to screen for AcIV-E var1 and var2 and can be used on a large scale in Europe to trace these two variants which likely represent two genetic lineages.