Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra‐segmental template switching recombination

Abstract

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.

First report of successful isolation of a HPR0‐like variant of the infectious salmon anaemia virus (ISAV) using cell culture

“…HPRΔ variants (deleted) can cause disease whereas HPR0 variants (no deletion) are not known to cause disease (Cook‐Versloot et al., 2004 ; Cunningham et al., 2002 ). With the exception of an artificial recombinant HPR0 variant (Cardenas et al., 2020 ), only HPRΔ variants are known to grow in cell culture (Aamelfot et al., 2016 ; OIE, 2021 ) and evidence suggests that HPRΔ originate from HPR0 variants, suggesting that transient variants with some but not all virulence markers may exist (Christiansen et al., 2017 ; Gagne & LeBlanc, 2018 ). On S5, a prerequisite of virulence seems to be a Q 266 →L 266 substitution or Q 266 with an insertion close to the putative cleavage site (Devold, 2006 ; Markussen et al., 2008 ).…”

Description of spatiotemporal patterns of infectious salmon anemia virus (ISAV) detections in marine Atlantic Salmon farms in Newfoundland and Labrador

“…However, in univariable analysis, TTD was much shorter for cases due to the ISAV-HPR0 variant, suggesting that fish are exposed much earlier during the production cycle with this virus variant, which likely supports the hypothesis that this is a circulating variant in the environment and could be the precursors of HPRΔ variants. The variant ISAV-HPR0 is considered a risk factor for developing clinical ISAV outbreaks as there is evidence for the conversion of ISAV-HPR0 into ISAV-HPRΔ in other regions (Cárdenas et al 2020). Our limited data did not provide any evidence to support this hypothesis.…”

“…The variant ISAV‐HPR0 is considered a risk factor for developing clinical ISAV outbreaks as there is evidence for the conversion of ISAV‐HPR0 into ISAV‐HPRΔ in other regions (Cárdenas et al. 2020). Our limited data did not provide any evidence to support this hypothesis.…”

Infectious Salmon Anemia Virus Infectivity Is Determined by Multiple Segments with an Important Contribution from Segment 5

“…Before the characterization of the infective phenotype, the synthetic reassortant viruses were genetically characterized to confirm the rearrangement and segments 5 and 6 genotype, since it has been shown that ISAV can modify its HPR region in vivo [ 16 ]. We have also described this phenomenon with our reverse genetics system in vitro [ 30 ]. However, we have demonstrated that it is possible to obtain genetically stable viruses via at least three passages with this system.…”

“…Subsequently, the supernatant was discarded, the cells were washed three times with PBS, and 1 mL of culture media with 10% FBS was added. The plates were maintained for seven days at 15 °C, and the process was repeated until obtaining three blind passages [ 30 ].…”