Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90-140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.

Abstract. Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15-20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (ϳ20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.

Abstract.We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P Ͻ 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R ϭ 0.81, 0.70, and 0.78, respectively; P Ͻ 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.Key words: Infectious salmon anemia (ISA); Infectious salmon anaemia virus (ISAV); ISAV virulence; ISAV pathogenesis; in situ hybridization; ISAV-in situ hybridization.Infectious salmon anemia (ISA) is a highly fatal disease of marine-farmed Atlantic salmon (Salmo salar L.) caused by ISA virus (ISAV), which belongs to the virus family Orthomyxoviridae 15,19,33,43 in the genus Isavirus. 2 Clinical disease is characterized by exophthalmia, pale gills, ascites, congestion of gut, enlargement of liver and spleen, petechial hemorrhages in the visceral organs, severe anemia, and variable mortality. 16,45 Histopathologic changes can include hepatic necrosis, vasculitis and sinusoidal congestion-peliosis, renal interstitial congestion, hemorrhage and tubular necrosis, congestion of branchial lamellar and filamental vessels, congestion and necrosis of the intestine and pyloric caeca, and increased erythrophagia in the spleen and kidney. 8,16,35,45 The disease, now a major threat to Atlantic salmon farming worldwide, was first recognized in Norway in 1984, 45 Canada in 1996, 6,30,35 Scotland in 1998 Faeroes Island, Denmark, in 2000, 1 and USA in 2001. 5 The virus has also been recovered from tissues of diseased farmed Coho salmon (Onchorynchus kisutch) in Chile. 25 The diagnosis of ISA has been based on clinical signs, histopathology, 45 virus isolation using different fish cell lines, 6,11,12,46 ...

ABSTRACT:In situ hybridization (ISH) was used to investigate the presence of viral mRNA in different fractions of blood cells of Atlantic salmon between 6 and 12 d following experimental infection with infectious salmon anemia virus (ISAV). Using a riboprobe targeting ISAV segment 7 mRNA, hybridization signals were observed only in leucocytes in buffy coat smears from samples collected from 8 to 12 d post-infection (dpi). None of the red blood cell smears from any sample showed ISH signal. These observations allow us to conclude that viremia in ISAV infection is associated with virus replication in leucocytes, and that erythrocytes are not target cells for virus replication. KEY WORDS: ISAV · Cell-associated viremia · In situ hybridization · Atlantic salmon Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [153][154][155][156][157] 2005 cides with the peak viremia period (Moneke et al. 2005). Previous in situ hybridization (ISH) studies on ISAV-infected fish have not reported on viral mRNA in blood cells (Gregory 2002, Moneke et al. 2003 although we have occasionally observed ISH signals in some circulating blood cells suspected as erythrocytes and in some individual cells in the head kidney suspected to be hematopoietic cells (Moneke et al. 2004). It was therefore decided to use ISH to more specifically identify the circulating blood cell(s) carrying virus during viremia. MATERIALS AND METHODS Cells and viruses.The ISAV isolate Norway 810/9/99 used for this study was propagated and titrated in the TO cell line as described previously (Kibenge et al. 2001).Riboprobe preparation. Preparation of the ISAV riboprobe was carried out as previously described (Moneke et al. 2003). Briefly, total RNA extracted from ISAV-infected cell culture lysate with Trizol Reagent (Invitrogen Life Technologies) was used in reverse transcription-polymerase chain reaction (RT-PCR) to obtain ISAV cDNA. The PCR primers consisted of ISAV RNA segment 7 (Ritchie et al. 2002, GenBank Acc. No. AF328627) forward primer 5'-ATG TCT GGA TTT AAC CTC GAG G-3' (nucleotides 133-154) and segment 7 reverse primer 5'-CAT AAC AAG TTT TCA ACC AAT C-3' (nucleotides 902-923) that yield a product 792 bp long. One-step RT-PCR was carried out as previously described (Kibenge et al. 2000), using Titan-one tube RT-PCR kit (Roche Molecular Biochemicals). The RT-PCR product was first cloned in the pCR ® II-TOPO ® Vector (Invitrogen Life Technologies) following the manufacturer's protocol. The insert DNA was then subcloned into pGEM-3Z vector (Promega) using the Rapid DNA Ligation Kit (Roche Molecular Biochemicals). The orientation of the DNA insert in the plasmid was determined by restriction enzyme analysis in order to allow generation off the T7 promoter, an antisense riboprobe of approximately 405 bases long. Thus the plasmid DNA was digested with Xba I prior to use. The in vitro transcription reaction was carried out in presence of digoxigenin-11-deoxyuridine triphosphate (Digoxigenin-11-UTP, Roche Molecular Bi...

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