Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes

<p>Abstract</p> <p>Background</p> <p>Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus <it>Isavirus</it>, family <it>Orthomyxoviridae</it>. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.</p> <p>Results</p> <p>Haemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID₅₀) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-α in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 – 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction.</p> <p>Conclusion</p> <p>ISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-α. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.</p

Effects of temperature and body size on immunological development and responsiveness in juvenile shortnose sturgeon (Acipenser brevirostrum)

Sturgeon are an important evolutionary taxa of which little is known regarding their responses to environmental factors. Water temperature strongly influences growth in fish; however, its effect on sturgeon immune responses is unknown. The objective of this study was to assess how 2 different temperatures affect immune responses in shortnose sturgeon (Acipenser brevirostrum) relevant immune organs such as the meningeal myeloid tissue, spleen, thymus and skin. These responses were studied in 2 different sizes of same age juvenile sturgeon kept at either 11 °C or 20 °C (4 treatment groups), before and after exposure to an ectoparasitic copepod (Dichelesthium oblongum). Based on a differential cell count, temperature was found to strongly influence immune cell production in the meningeal myeloid tissue, regardless of the fish sizes considered. Morphometric analysis of splenic white pulp showed a transient response to temperature. There were no differences between the groups in the morphometric analysis of thymus size. Splenic IRF-1 and IRF-2 had similar expression profiles, significantly higher in fish kept at 20 °C for the first 6 weeks of the study but not by 14 weeks. In the skin, IRF-1 was significantly higher in the fish kept at 11 °C over the first 6 weeks of the study. IRF-2 had a similar profile but there were no differences between the groups by the end of the trial. In conclusion, higher water temperatures (up to 20 °C) may have beneficial effects in maximizing growth and improving immunological capacity, regardless of the fish sizes considered in this study

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes-1

present; i.e., in response to L-15 medium alone (negative control) calibrated to the 18S rRNA housekeeping gene and the 0 hour control (values are average of a triplicate observation ± standard deviation): Atlantic salmon IFN genes (SasaIFN-α1 and SasaIFN-α2), Mx, ISG15 and STAT1 in negative control erythrocytes.<p><b>Copyright information:</b></p><p>Taken from "Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes"</p><p>http://www.virologyj.com/content/5/1/36</p><p>Virology Journal 2008;5():36-36.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2292172.</p><p></p

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes-3

18S rRNA housekeeping gene and the 0 hour control (values are average of a triplicate observation ± standard deviation).<p><b>Copyright information:</b></p><p>Taken from "Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes"</p><p>http://www.virologyj.com/content/5/1/36</p><p>Virology Journal 2008;5():36-36.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2292172.</p><p></p

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes-0

Ination calibrated to the 18S rRNA housekeeping gene and the 0 hour control (values are average of a triplicate observation ± standard deviation): relative fold stimulation of Atlantic salmon IFN genes (SasaIFN-α1 and SasaIFN-α2), Mx, ISG15 and STAT1 by NBISA01 virus; relative fold stimulation of Atlantic salmon IFN genes (SasaIFN-α1 and SasaIFN-α2), Mx, ISG15 and STAT1 by RPC/NB-04-085-1.<p><b>Copyright information:</b></p><p>Taken from "Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes"</p><p>http://www.virologyj.com/content/5/1/36</p><p>Virology Journal 2008;5():36-36.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2292172.</p><p></p

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes-2

0 hour control (values are average of a triplicate observation ± standard deviation): UV-inactivated NBISA01; heat-inactivated NBISA01; UV-inactivated RPC/NB-04-085-1; and heat inactivated RPC/NB-04-085-1 haemagglutination.<p><b>Copyright information:</b></p><p>Taken from "Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes"</p><p>http://www.virologyj.com/content/5/1/36</p><p>Virology Journal 2008;5():36-36.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2292172.</p><p></p