First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Abstract

Background: Infectious salmon anaemia (ISA) is a viral disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. The virus is considered to be carried by marine wild fish and for over 25 years has caused major disease outbreaks in marine-farmed Atlantic salmon in the Northern hemisphere. In the Southern hemisphere, ISAV was first detected in Chile in 1999 in marine-farmed Coho salmon (Oncorhynchus kisutch). In contrast to the classical presentation of ISA in Atlantic salmon, the presence of ISAV in Chile until now has only been associated with a clinical condition called Icterus Syndrome in Coho salmon and virus isolation has not always been possible. During the winter of 2007, unexplained mortalities were registered in market-size Atlantic salmon in a grow-out site located in Chiloé in Region X of Chile. We report here the diagnostic findings of the first significant clinical outbreak of ISA in marine-farmed Atlantic salmon in Chile and the first characterization of the ISAV isolated from the affected fish.

Results: In mid-June 2007, an Atlantic salmon marine farm site located in central Chiloé Island in Region X of Chile registered a sudden increase in mortality following recovery from an outbreak of Pisciricketsiosis, which rose to a cumulative mortality of 13.6% by harvest time. Based on the clinical signs and lesions in the affected fish, and laboratory tests performed on the fish tissues, a confirmatory diagnosis of ISA was made; the first time ISA in its classical presentation and for the first time affecting farmed Atlantic salmon in Chile. Rapid sequencing of the virus-specific RT-PCR products amplified from the fish tissues identified the virus to belong to the European genotype (Genotype I) of the highly polymorphic region (HPR) group HPR 7b, but with an 11-amino acid insert in the fusion glycoprotein, and ability to cause cytopathic effects (CPE) in CHSE-214 cell line, characteristics which make it distinct from common European Genotype ISAV isolates from Europe and North America.

Conclusion: In conclusion, the present work constitutes the first report of a case of ISA in farmed Atlantic salmon in Chile. The clinical signs and lesions are consistent with the classical descriptions of the disease in marine-farmed Atlantic salmon in the Northern hemisphere. The outbreak was caused by ISAV of European genotype (or Genotype I) of HPR 7b but distinct from common European Genotype ISAV isolates.

Figure 1

Geographical location and mortality pattern of the first ISA outbreak in Chile. (A)Geographical location of the Atlantic salmon marine farm site: Lemuy Island, Central Chiloé, Region X, Chile. (B)Weekly percent cummulative mortality up to the time of total harvest of the Atlantic salmon marine farm with ISA outbreak. Net Pen A and Net Pen B represent mortality in the 2 cages with ISA. FARM corresponds to the total mortality on the farm up to the time of harvest.

Figure 2

Gross lesions in affected Atlantic salmon from the ISA outbreak. (A) Frequency of the external gross lesions in affected Atlantic salmon from the ISA outbreak. Percentage of fish with a specified lesion among 100 fish necropsied. (B) Common gross lesions seen at necropsy: Top panel – Atlantic salmon with exophthalmia and pale gills. Middle panel – Atlantic salmon with petechial haemorrhages on the abdomen. Bottom panel – Atlantic salmon with very dark liver and haemorrhages on the visceral adipose tissue. (C) Frequency of the internal gross lesions in affected Atlantic salmon from the ISA outbreak. Percentage of fish with a specified lesion among 100 fish necropsied.

Figure 3

Microscopic lesions in affected Atlantic salmon from the ISA outbreak. (A) Histologic section of intestine of Atlantic salmon. H&E staining. (→) indicates mucosal ulceration and haemorrhage. (B) Histologic section of liver of Atlantic salmon from the ISA outbreak H&E staining. (→) indicates hepatocellular necrosis, with marked haemorrhage and sinusoidal congestion/peliosis. (C) Histologic section of spleen of Atlantic salmon from the ISA outbreak H&E staining. (→) indicates endothelial erythrophagia.

Figure 4

ISAV antigen staining of samples from the ISA outbreak. (A) Immunohistochemical staining of histologic section of heart of Atlantic salmon from the ISA outbreak. Dark brown colour indicates positive staining in endothelial cells with anti-ISAV monoclonal antibody. (B)Virus isolation in CHSE-214 cells: ISAV-infected cells (250×) showing fluorescent staining following IFAT with anti-ISAV monoclonal antibody.

Figure 5

Alignment of amino acid sequences in the proteolytic cleavage site of the precursor F₀ protein (modified from Kibenge et al. [19]). The amino acid sequence corresponding to the fusion protein of the Chilean ISAV in this disease outbreak is highlighted in yellow. The designation of amino acid inserts IN1, IN2, and IN3 are as reported by Devold et al. [21]. The unique 11-amino acid insert found in the Chilean ISAV in this disease outbreak is designated IN4. Other sources of information are indicated as *Devold et al. [21], **Markussen et al. [20], and ***Plarre and Nylund (2004; SF83/04, GenBank Accession No. AY744392). It has been suggested by Markussen et al. [20] that ISAV isolate SF83/04 represents a mixed virus infection of HPR0 and another HPR group, which might explain the confusion in the virulence designation.

Figure 6

Alignment of amino acid sequences in the highly polymorphic region (HPR) of the HE genes of various strains of ISAV (modified from Kibenge et al. [19]). The amino acid sequence corresponding to the HE-HPR of the Chilean ISAV in this disease outbreak is highlighted in yellow, and identifies it as HPR7. Sequences that are not determined are indicated by dots, and amino acid (aa) deletions in the HPR are indicated by dashes. The HPR groups identified are as reported by Nylund et al. [23] and Plarre et al. [24]; other sources of information are indicated as *Markussen et al. [20], **Cunningham et al. [25], ***Cook-Versloot et al. [26], and ****Mjaaland et al. [27].