Infectious Salmon Anemia Virus RNA in Fish Cell Cultures and in Tissue Sections of Atlantic Salmon Experimentally Infected with Infectious Salmon Anemia Virus

Abstract

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.

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

“…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.…”

“…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.…”

“…ISH was performed on the blood smears using the ISAV RNA segment 7 riboprobe following the procedure used for fish tissues (Moneke et al 2003). Briefly, the blood smears were rehydrated in decreasing concentrations of ethanol to a final wash in diethyl pyrocarbonate (DEPC)-treated.…”

“…The cells were permeabilized and then hybridized with approximately 25 ng of the riboprobe in hybridization buffer per slide. ISH signals were developed using the Dig-UTP-Detection kit (Roche Diagnos-tics); they were then observed using light microscopy and the images captured digitally were enhanced and analyzed using Adobe Photoshop as previously described (Moneke et al 2003).…”

“…The digoxigenin-labeled RNA transcripts were purified by ethanol precipitation in high salt and were stored at -80°C until used. The integrity, polarity, specificity and sensitivity of the riboprobe were checked by Northern blot hybridization as previously described (Moneke et al 2003). …”