Genetic analysis and comparative virulence of infectious salmon anemia virus (ISAV) types HPR7a and HPR7b from recent field outbreaks in Chile

Infectious salmon anemia (ISA) is a serious viral disease of marine farmed Atlantic salmon, Salmo salar L. caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae[1]. The mortality in marine fish net-cages rises slowly and can vary from 0 to 90%. In fact, the virus can be present in the net-cage for up to 6 months before significant mortality is noted. ISA is arguably the most important viral disease of marine-farmed Atlantic salmon because of the associated socio-economic losses, and ISAV remains an emerging fish pathogen because of the asymptomatic infections in wild and farmed fish and the potential for emergence of new epizootic strains.

The ISAV genome consists of eight single-stranded RNA segments [1]; sequence analysis of these segments from different ISAV isolates consistently reveals two genotypes designated according to their geographic origin as European (Genotype I) and North American (Genotype II) [2]. Genotype I can be further subdivided into Genogroup 1 (European–in-North America, EU-in-NA) for Genotype I strains also found in North America, and Genogroup 2 (Real European, Real-EU) for Genotype I strains only found in Europe and Chile [3]. ISAV of European genotype can also be differentiated into three genogroups, EU-G1, EU-G2, and EU-G3; the EU-in-NA strains are placed in EU-G2 group, and Real European strains are divided between EU-G1 and EU-G3 groups [4].

ISAV genomic segments 5 and 6 encode two major surface glycoproteins that are believed to be important for the pathogenicity of ISAV. Hemagglutinin-esterase (HE) encoded by segment 6 has receptor-destroying activity and is responsible for receptor binding and virus release [5]-[7], while the fusion (F) protein encoded by segment 5 is responsible for the fusion of the viral and cellular membranes during virus entry [8]. The systemic ISA is caused by virulent ISAV strains with deletions in HE hyper polymorphic region (HPR) spanning residues V³³⁷ to M³⁷² in the stem adjacent to the transmembrane portion of the protein; the virus is therefore designated as ISAV-HPRΔ. For both European and North American genotypes, a direct functional relationship can be demonstrated between the length of HE protein stem, ISAV cytopathogenicity in cell culture, and pathogenicity for Atlantic salmon [3]. All ISAV-HPRΔ isolates also have either a sequence insertion or Q²⁶⁶ → L²⁶⁶ mutation in the F protein [3],[9],[10]. A single report of ISAV-HPR7b with Q²⁶⁶ in the F protein provided no sequence data to authenticate it [11]. To date, four types of sequence insertions (IN1 to IN4) have been reported in the F protein: IN1 to IN3 have been detected in Norwegian isolates and IN4 only in Chilean isolates [3].

Non-cultivable, non-pathogenic ISAV detectable only by RT-PCR has full-length HPR sequence (35 amino acids) in the HE-encoding gene and is designated ISAV-HPR0 [4],[10],[12]-[19]. All ISAV-HPR0 isolates are characterized by the wild-type F protein with Q²⁶⁶ and no sequence insertion [10]. ISAV-HPR0 replicates mainly in the gills causing only transient subclinical infection [16]. This virus has been detected in apparently healthy wild and farmed Atlantic salmon in most regions with Atlantic salmon aquaculture [20], and is considered to have ancestral relationship with ISAV-HPRΔ [21]. Most recently, ISA cases directly linked to the presence of endemic ISAV-HPR0 have been reported [20], supporting the notion that under appropriate conditions ISAV-HPR0 can mutate to ISAV-HPRΔ.

In Chile, the first ISA outbreak in marine-farmed Atlantic salmon in mid-June 2007 was officially confirmed to be associated with ISAV-HPR7b, which was similar to Norwegian isolates, but had acquired an insertion of 33 base pairs in segment 5 [3]. Phylogenetic analysis of ISAV isolates from different outbreaks suggested that the virus was introduced from Norway in 1996 [3], probably through fertilized salmon eggs [22],[23].

Since 2010, ISAV-HPR0 has been detected in Chile in Atlantic salmon at different production stages without any clinical signs of ISA until April 2013, when the re-emerged ISAV-HPRΔ (ISAV-HPR3 and ISAV-HPR14) caused two ISA outbreaks in two marine Atlantic salmon farms [20]. Prior to these outbreaks, the predominant strain was ISAV-HPR7b, which comprised 79% of ISAV isolates from 2007–2010 ISA outbreaks [3]. Phylogenetically, Chilean isolates ISAV-HPR3 and ISAV-HPR14 clustered with ISAV-HPR0, also detected in Chile [20]. ISAV-HPR14 had the motif ³⁶⁰PAT³⁶² characteristic of Chilean ISAV-HPR0 variant [20]. Subsequently, new ISA outbreaks caused by ISAV-HPR7a and ISAV-HPR7b were recorded in November 2013 and January 2014, respectively. These ISAV isolates are different from the ones previously reported in Virol J 2013, 10:344 [20]. In the present study, we describe the genetic characteristics and relationship between ISAV-HPR7a and ISAV-HPR7b that caused the ISA outbreaks in Chile in 2013 and 2014, and the evolution of the ISAV clades since our original analysis in 2009.

The last ISA outbreak in 2013 in Chile was attributed to ISAV-HPR7a; this virus did not have a sequence insertion in segment 5 when sequenced directly from salmon tissues, but had this insertion when the virus isolated from cell culture was sequenced. The first ISA outbreak reported in 2014 in Chile was associated with ISAV-HPR7b without insertion in segment 5. Both viruses had a 23-amino acid deletion in HPR of segment 6, but differed in that HPR7a had threonine and HPR7b had isoleucine at position 370. In segment 5, the salmon tissue-originated ISAV-HPR7a had Q²⁶⁶ → L²⁶⁶ substitution, whereas the cell culture-grown ISAV-HPR7a had an 18-amino acid insertion LEVVREKGDDTSQSDSFY of 100% sequence identity with ISAV RNA segment 1 encoding PB2 polymerase. These data indicate the variability in the two ISAV virulence markers, especially in case of the ISAV-HPR7a isolate, depending on virus growth conditions (salmon tissue or cell culture). The virulent ISAV-HPRΔ strains have deletion in HPR as well as Q²⁶⁶ → L²⁶⁶ substitution or insertion in the putative proteolytic site of the F protein [10]. The last ISA outbreak of 2010 was caused by ISAV-HPR7b strains with and without insertion in segment 5, but none of them was similar to the ISAV-HPR7b isolate responsible for ISA outbreak in 2007, which had an 11-amino acid insertion of 100% sequence identity with ISAV RNA segment 2 in the F protein [3]. This insertion might have occurred through non-homologous recombination between the F- and PB1-encoding genes of the same virus; more than 85% of the outbreak cases were caused by this virus [3]. The last ISA outbreaks in Chile confirm the variability in ISAV virulence markers because the 2013 cases were associated with ISAV-HPR3 and ISAV-HPR14 without sequence insertions in segment 5 [20], supporting the quasispecies hypothesis, which suggests that multiple evolutionary forces account for ISAV genetic diversity [3]. The ISAV virulence in the six cases in this study was different too: ISAV-HPR7a appeared to be more virulent than ISAV-HPR7b without sequence insertion in segment 5 (MGG, personal communication). It is therefore possible that the sequence insertion in segment 5 of the 2007 ISAV-HPR7b isolate contributed to the virulence of the virus.

The virulent ISAV isolates in Chile show a close relationship with ISAV-HPR0 [20]; by 2012, the low-pathogenic ISAV-HPR0 strain had completely replaced the virulent ISAV-HPR∆ strain responsible for the 2007–2010 ISA outbreaks, as the dominant ISAV variant in marine-farmed Atlantic salmon in Chile. The occurrence of two new ISA outbreaks in April 2013 marked a brief re-emergence of virulent ISAV-HPR∆, demonstrating that ISAV-HPR0 dissemination together with the sporadic ISAV-HPR∆ returns is a characteristic feature of ISA post-crisis dynamics [12],[14],[16],[17].

Based on our understanding about virus transmissions where a virus transmission occurrence can be roughly classified into the latent stage and the breakout stage; and mutation rates tend to be slow in the latent stage and fast in the breakout stage, the variable mutation rate model used in Figure 2 is consistent with a typical virus transmission. Typically, a small amount of virus is physically transmitted to a new location and finds suitable hosts and the latent stage starts. In this stage, mutation occurs at a slow rate and in a unified way. When the amount of virus accumulates to a certain level, the breakout stage starts and the mutation rate is faster. ISAV isolates from Norway transmitted to Chile around 1996 might have existed in the same or different locations in Chile and gone through different evolution pathways: Chile 1 entered the breakout stage early around 2005; Chile 2 entered the breakout stage at some time before 2013; and Chile 3 might still be in the latent stage. The phylogenetic tree of the concatenated segment 5 and 6 sequence (Figure 2) confirms that the 1996 divergence point is still evident in the Chilean ISAV isolates.

In summary, in the last two to three years, we observed active evolution of Chilean ISAV isolates. The samples collected in 2013 and 2014 indicate that ISAV has evolved to an extent that one new group (Chile 2) has already been confirmed and another new group (Chile 3) has emerged and will probably be recognized as such in the near future.