Morphology and morphogenesis of infectious salmon anaemia virus replicating in the endothelium of Atlantic salmon Salmo salar

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...

Section: Resultssupporting

confidence: 81%

Viremia during infectious salmon anemia virus infection of Atlantic salmon is associated with replicating virus in leucocytes

Moneke¹,

Ikede²,

Kibenge³

2005

“…In contrast to orthomyxovirus infections in mammals, ISAV infection is not restricted to the respiratory system. By electronmicroscopy, budding of ISAV has been localised to endothelial cells of blood vessels in several organs , Koren & Nylund 1997. Endothelial cells may therefore be target cells for ISAV.…”

Section: Discussionmentioning

confidence: 99%

Time course tissue distribution of infectious salmon anaemia virus in experimentally infected Atlantic salmon Salmo salar

Rimstad

Falk²,

Mikalsen³

et al. 1999

Atlantic salmon Salmo salar L. were injected intrapentoneally with infectious salmon anaemia virus (1SAV)-infective tissue homogenate to clarify the tissue distribution of ISAV in a time course study. Fish were sampled at 11 different intervals between 1 and 40 d post-infection (p.i.) and mid-kidney, head kidney, liver, spleen, intestine, gills, muscle and heart were tested for the presence of ISAV by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that during a disease outbreak, ISAV is present in most organs. It was possible to detect ISAV at all sampling times in at least 1 of the fish examined. However, for the first 8 d p.i. positive RT-PCR results were predominantly found in samples from the head hdney and mid-kidney. Fish giving positive samples after Day 13 p.i. were RT-PCR positive in most organs. These results indicated that between Days 8 to 13 p.i. considerable replication of the virus occurred, combined with wide tissue dissemination.

“…ISA has also been reported from farmed Atlantic salmon in Scotland, Canada, the Faroe Islands and the USA (Mullins et al 1998, Rodger et al 1998, Bouchard et al 2001. The viral agent is an enveloped, negativestranded, RNA virus closely resembling orthomyxoviruses , Koren & Nylund 1997, Krøssoy et al 1999. ISA is characterised by several haematological abnormalities including severe anaemia, ascites, congestion and enlargement of the liver and spleen, congestion in the intestinal mucosa, petechiae in the visceral fat, and high mortality (Koren & Nylund 1997).…”

Section: Introductionmentioning

confidence: 99%

“…The viral agent is an enveloped, negativestranded, RNA virus closely resembling orthomyxoviruses , Koren & Nylund 1997, Krøssoy et al 1999. ISA is characterised by several haematological abnormalities including severe anaemia, ascites, congestion and enlargement of the liver and spleen, congestion in the intestinal mucosa, petechiae in the visceral fat, and high mortality (Koren & Nylund 1997). However, the ISA virus may also be present without causing any mortality in salmon (Nylund pers.obs.…”

Section: Introductionmentioning

confidence: 99%

Cardiac performance in Salmo salar with infectious salmon anaemia (ISA): putative role of nitric oxide

Gattuso¹,

Mazza²,

Imbrogno³

et al. 2002

Self Cite

Infectious salmon anaemia (ISA) is a viral disease which targets the vascular and endocardial endothelial cells. An in vitro preparation of the spontaneously beating working heart of Salmo salar L. (i.e. able to generate physiological values of output pressure, cardiac output, ventricle work and power) was used to study cardiac performance under basal (i.e. in the absence of stimuli) and loading (i.e. Frank-Starling response) conditions in both control and ISAV-affected fish. In contrast to control fish, the heart preparations of the infected counterparts showed an impairment of the FrankStarling response, particularly evident in fish infected with a higher virus dose. The Frank-Starling response was progressively impaired with the progression of the viral disease from the time of the virus administration until the 20th day. The potential involvement of nitric oxide (NO) in cardiac dysfunction was investigated by using the authentic nitric oxide synthase (NOS) substrate L-arginine and the NOS inhibitors L-NMMA and L-NIL. In contrast to control fish, infected hearts were particularly sensitive to the inducible nitric oxide synthase (iNOS) inhibitor L-NIL and insensitive to L-arginine. While pretreatment with NOS inhibitors reduced the Frank-Starling response in control hearts, it restored this response in infected counterparts. Taken together, the results indicate that cardiac dysfunction and the NO-transduction-pathway can be mechanistically linked in infected salmon.KEY WORDS: ISA virus · Salmon · Heart · Nitric oxide · Myocardial dysfunction Resale or republication not permitted without written consent of the publisherDis Aquat Org 52: [11][12][13][14][15][16][17][18][19][20] 2002 from polymorphonuclear leukocytes shows that these cells may also be target cells . In these cell types, which are metabolically active and capable of phagocytosis, the ISA virus is able to replicate. Indeed, from these cells the virus particles can frequently be observed budding in the lumen of the heart and the blood vessels (Nylund et al. , 1996.The autocrine-paracrine function of the Ev and EE, which involves the synthesis and release of nitric oxide (NO), in addition to endothelin, prostacyclin and other autacoids, is well recognised. In particular, Ev and EE, in addition to cardiomyocytes, blood platelets, and other cell types, represent the predominant source of one of the 3 NOS isoforms, the 'endothelial' NOS (eNOS), so named as it is isolated, purified and cloned from the vascular endothelium (see e.g. Andries et al. 1998, Busse & Fleming 2000. The expression and the activity of this isoform, with the consequent production of NO, plays a major role in controlling various functions of the peripheral vasculature (Busse & Fleming 2000) and the heart (Balligand 2000). After induction by inflammatory and immunologic stimuli, including a diversity of infectious agents, the Ev and EE, like most cell types such as macrophages, cardiac myocytes, vascular smooth muscle cells, glial cells (to name only a few), express al...