The ISAV has a genome composed of eight segments of (–)ssRNA, segment 6 codes for the hemagglutinin–esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6‐HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra‐segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Infectious salmon anemia virus (ISAV) is the etiological agent of infectious salmon anemia. It belongs to the genus isavirus, one of the genera of the Orthomyxoviridae family, as does Influenzavirus A. The ISAV genome comprises eight negative-sense single-stranded RNA segments that code for at least 10 proteins. Although some ISAV strains can reach 100% mortality rates, the factors that determine isavirus infectivity remain unknown. However, some studies suggest that segments 5 and 6 are responsible for the different degrees of virulence and infectivity among ISAV subtypes, unlike the influenza A virus, where most segments are involved in the virus infectivity. In this work, synthetic reassortant viruses for the eight segments of ISAV were generated by reverse genetics, combining a highly virulent virus, ISAV 752_09 (HPR7b), and an avirulent strain, SK779/06 (HPR0). We characterized the rescued viruses and their capacity to replicate and infect different cell lines, produce plaques in ASK cells, and their ability to induce and modulate the cellular immune response in vitro. Our results show that the majority of ISAV segments are involved in at least one of the analyzed characteristics, segment 5 being one of the most important, allowing HPR0 viruses, among other things, to produce plaques and replicate in CHSE-214 cells. We determined that segments 5 and 6 participate in different stages of the viral cycle, and their compatibility is critical for viral infection. Additionally, we demonstrated that segment 2 can modulate the cellular immune response. Our results indicate a high degree of genetic compatibility between the genomic segments of HPR7b and HPR0, representing a latent risk of reassortant that would give rise to a new virus with an unknown phenotype.