Early detection of salmonid alphavirus in seawater from marine farm sites of Atlantic salmon Salmo salar.

The traditional strategy for national surveillance of salmonid alphavirus (SAV) infection in Norwegian fish farms relies on a costly, time-consuming, and resource-demanding approach based on the monthly sampling of fish from all marine farms with salmonids. In order to develop an alternative surveillance method, a water filtration method was tested in parallel with the ongoing surveillance program at 7 Norwegian marine farm sites of Atlantic salmon Salmo salar L. with no current suspicion of SAV infection. During the period from May 2019 to January 2020, seawater samples were collected from the top layer water inside all net-pens at these 7 sites. The samples were concentrated for SAV by filtration through an MF-Millipore™ electronegative membrane filter, followed by rinsing with NucliSENS® Lysis Buffer, before RNA extraction and analysis by RT-qPCR. SAV was detected from seawater at an earlier stage compared to traditional sampling methods, at all sites where the fish tested positive for SAV. A significant negative relationship was observed at all sites between the SAV concentration found in seawater samples and the number of days until SAV was detected in the fish. This means that the fewer the SAV particles in the seawater, the more days it took until SAV was detected in the fish samples. Based on this, sampling of seawater every month for the surveillance of SAV has a great potential as an alternative method for early detection of SAV in Atlantic salmon farms.

The in situ distribution of glycoprotein-bound 4-O-Acetylated sialic acids in vertebrates.

Sialic acids are located at the terminal branches of the cell glycocalyx and secreted glycan molecules. O-Acetylation is an important modification of the sialic acids, however very few studies have demonstrated the in situ distribution of the O-Acetylated sialic acids. Here the distribution of glycoprotein bound 4-O-Acetylated sialic acids (4-O-Ac sias) in vertebrates was determined using a novel virus histochemistry assay. The 4-O-Ac sias were found in the circulatory system, i.e. on the surface of endothelial cells and RBCs, of several vertebrate species, though most frequently in the cartilaginous fish (class Chondrichthyes) and the bony fish (class Osteichthyes). The O-Acetylated sialic acid was detected in 64 % of the examined fish species. Even though the sialic acid was found less commonly in higher vertebrates, it was found at the same location in the positive species. The general significance of this endothelial labelling pattern distribution is discussed. The seemingly conserved local position through the evolution of the vertebrates, suggests an evolutionary advantage of this sialic acid modification.

A refinement to eRNA and eDNA-based detection methods for reliable and cost-efficient screening of pathogens in Atlantic salmon aquaculture.

Finfish aquaculture is one of the fastest-growing food production sectors in the world, and numerous infectious diseases are a constant challenge to the fish farming industry, causing decreased fish health and, consequently, economic losses. Specific and sensitive tools for pathogen detection are crucial for the surveillance of environmental samples to prevent the spread of fish pathogens in farms. Monitoring of waterborne pathogens through filtration of water and subsequent molecular detection of target-specific DNA or RNA sequence motifs is an animal-friendly method. This approach could reduce or even replace the sacrifice of fish for monitoring purposes in aquaculture and allow earlier implementation of disease control measures. Sampling methods might be a bottleneck, and there is a need for simple sampling methods that still ensure the best detection probability. In this study, we tested different filtration methods with spiked freshwater and seawater for a panel of fish pathogens to discern a suitable procedure that can be easily applied on-site by farm personnel without compromising detection probability. Specifically, we tested combinations of different filtration flow rates, lysis buffers, and filters for the detection of some of the pathogens relevant to the aquaculture industry. The results showed that a "sandwich" filtration method using two different filters and a flow rate of up to 4.0 L/min ensured good pathogen detection. The filters, consisting of a hydrophilic glass fibre filter with binder resin on the top and a hydrophilic mixed cellulose esters membrane at the bottom, achieved the best concentration and qPCR detection of both viral and bacterial fish pathogens. This up-and-coming tool allows the detection of very different fish pathogens during a single filtration step, and it can be combined with one single automated total nucleic acid extraction step for all the investigated pathogens, reducing both analysis costs and time.

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus.

Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.

Infectious salmon anaemia virus infection of Atlantic salmon gill epithelial cells.

Infectious salmon anaemia virus (ISAV), a member of the Orthomyxoviridae family, infects and causes disease in farmed Atlantic salmon (Salmo salar L.). Previous studies have shown Atlantic salmon endothelial cells to be the primary targets of ISAV infection. However, it is not known if cells other than endothelial cells play a role in ISAV tropism. To further assess cell tropism, we examined ISAV infection of Atlantic salmon gill epithelial cells in vivo and in vitro. We demonstrated the susceptibility of epithelial cells to ISAV infection. On comparison of primary gill epithelial cell cultures with ISAV permissive fish cell cultures, we found the virus yield in primary gill epithelial cells to be comparable with that of salmon head kidney (SHK)-1 cells, but lower than TO or Atlantic salmon kidney (ASK)-II cells. Light and transmission electron microscopy (TEM) revealed that the primary gill cells possessed characteristics consistent with epithelial cells. Virus histochemistry showed that gill epithelial cells expressed 4-O-acetylated sialic acid which is recognized as the ISAV receptor. To the best of our knowledge, this is the first demonstration of ISAV infection in Atlantic salmon primary gill epithelial cells. This study thus broadens our understanding of cell tropism and transmission of ISAV in Atlantic salmon.

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition.

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.

A sequential study of incomplete Freund's adjuvant-induced peritonitis in Atlantic cod.

Development of diagnostic and prophylactic methodologies is dependent on knowledge of the host's defence system and reaction to different vaccine adjuvants. Here we present a sequential morphological study of peritonitis and inflammatory cell processing of incomplete Freund's adjuvant (IFA) in intraperitoneally injected Atlantic cod. The peritoneal tissue responses were characterised using necropsy, histology and electron microscopy. An extensive inflammatory response as characterised by leukocyte morphology and contents of enzymes, presence of apoptotic cells and IFN-γ-expressing cells was observed. Three days post injection, IFA droplets were surrounded by different types of inflammatory cells and two different patterns could be discerned. The first was characterised by flattened and concentrically arranged interdigitating cells connected by desmosomes and with macrophage-like cells (MLCs) predominant in the periphery. The second type possessed four stratified layers with an inner layer containing many apoptotic MLCs; a second layer containing flattened and shrunken cells and outer layers comprising moderately flattened cells and an outermost layer of mononuclear cells expressing IFN-γ. Oil was detected both inside and outside MLCs. The two types of processes, of which the second was clearly stratified, were similar to those observed in other teleosts, indicating a variety of reaction modes or alternatively sequential process development. The numerous dead MLCs contributed to inflammation.

Salmon Erythrocytes Sequester Active Virus Particles in Infectious Salmon Anaemia.

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

Short communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater.

Infectious salmon anaemia virus (ISAV) is the cause of an important waterborne disease of farmed Atlantic salmon. Detection of virus in water samples may constitute an alternative method to sacrificing fish for surveillance of fish populations for the presence of ISA-virus. We aimed to evaluate different membrane filters and buffers for concentration and recovery of ISAV in seawater, prior to molecular detection. One litre each of artificial and natural seawater was spiked with ISAV, followed by concentration with different filters and subsequent elution with different buffers. The negatively charged MF hydrophilic membrane filter, combined with NucliSENS® lysis buffer, presented the highest ISAV recovery percentages with 12.5 ± 1.3% by RT-qPCR and 31.7 ± 10.7% by RT-ddPCR. For the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter, combined with NucliSENS® lysis buffer, the ISAV recovery percentages were 3.4 ± 0.1% by RT-qPCR and 10.8 ± 14.2% by RT-ddPCR. The limits of quantification (LOQ) were estimated to be 2.2 x 103 ISAV copies/L of natural seawater for both RT-qPCR and RT-ddPCR. The ISAV concentration method was more efficient in natural seawater.

Infectious Salmon Anemia Virus Shedding from Infected Atlantic Salmon (Salmo salar L.)-Application of a Droplet Digital PCR Assay for Virus Quantification in Seawater.

Infectious salmon anemia virus (ISAV) infection is currently detected by fish sampling for PCR and immunohistochemistry analysis. As an alternative to sampling fish, we evaluated two different membrane filters in combination with four buffers for elution, concentration, and detection of ISAV in seawater, during a bath challenge of Atlantic salmon (Salmo salar L.) post-smolts with high and low concentrations of ISAV. Transmission of ISAV in the bath challenge was confirmed by a high mortality, clinical signs associated with ISA disease, and detection of ISAV RNA in organ tissues and seawater samples. The electronegatively charged filter, combined with lysis buffer, gave significantly higher ISAV RNA detection by droplet digital PCR from seawater (5.6 × 104 ISAV RNA copies/L; p < 0.001). Viral shedding in seawater was first detected at two days post-challenge and peaked on day 11 post-challenge, one day before mortalities started in fish challenged with high dose ISAV, demonstrating that a large viral shedding event occurs before death. These data provide important information for ISAV shedding that is relevant for the development of improved surveillance tools based on water samples, transmission models, and management of ISA.

Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater.

Waterborne viral infections represent a major threat to fish health. For many viruses, understanding the interplay between pathogens, host and environment presents a major hurdle for transmission. Salmonid alphavirus (SAV) can infect and cause pancreas disease (PD) in farmed salmonids in seawater. During infection, SAV is excreted from infected fish to the seawater. We evaluated two types of filters and four different eluents, for concentration of SAV3. One L of seawater was spiked with SAV3, followed by filtration and virus elution from membrane filters. For the negatively charged MF hydrophilic membrane filter (MF-) combined with NucliSENS® lysis buffer the SAV3 recovery was 39.5 ±â€¯1.8 % by RT-ddPCR and 25.9 ±â€¯5.7 % by RT-qPCR. The recovery using the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter (MD+), combined with NucliSENS® lysis buffer was 19.0 ±â€¯0.1 % by RT-ddPCR and 13.3 ±â€¯3.8 % by RT-qPCR. The limits of quantification (LOQ) and detection (LOD) were estimated to be 5.18 × 103 and 2.0 × 102 SAV3 copies/L of natural seawater, by RT-ddPCR. SAV3 recovery from small volumes of seawater, and the requirement for standard laboratory equipment, suggest the MF-filter combined with NucliSENS® lysis buffer would be a candidate for further validation in experimental trials.

A case study of Desmozoon lepeophtherii infection in farmed Atlantic salmon associated with gill disease, peritonitis, intestinal infection, stunted growth, and increased mortality.

BACKGROUND: In September 2008, a disease outbreak characterized by acute, severe gill pathology and peritonitis, involving the gastrointestinal tract, was observed in an Atlantic salmon (Salmo salar L.) farm in north-western Norway. During subsequent sampling in November 2008 and January 2009, chronic proliferative gill inflammation and peritonitis was observed. Cumulative mortalities of 5.6-12.8% and severe growth retardation were observed. Routine diagnostic analysis revealed no diseases known to salmon at the time, but microsporidian infection of tissues was observed. METHODS: To characterize the disease outbreak, a combination of histopathology, in situ hybridization (ISH), chitin, calcofluor-white (CFW) staining, and real-time PCR were used to describe the disease progression with visualization of the D. lepeophtherii stages in situ. RESULTS: The presence of the microsporidian Desmozoon lepeophtherii was confirmed with real-time PCR, DNA sequencing and ISH, and the parasite was detected in association with acute lesions in the gills and peritoneum. ISH using a probe specific to small subunit 16S rRNA gene provided an effective tool for demonstrating the distribution of D. lepeophtherii in the tissue. Infection in the peritoneum seemed localized in and around pre-existing vaccine granulomas, and in the gastrointestinal walls. In the heart, kidney and spleen, the infection was most often associated with mononuclear leucocytes and macrophages, including melanomacrophages. Desmozoon lepeophtherii exospores were found in the nuclei of the gastrointestinal epithelium for the first time, suggesting a role of the gastrointestinal tract in the spread of spores to the environment. CONCLUSIONS: This study describes the progression of D. lepeophtherii disease outbreak in an Atlantic salmon farm without any other known diseases present. Using different methods to examine the disease outbreak, new insight into the pathology of D. lepeophtherii was obtained. The parasite was localized in situ in association with severe tissue damage and inflammation in the gills, peritoneal cavity and in the gastrointestinal (GI) tract that links the parasite directly to the observed pathology.

Avipoxvirus multiplication in a mammalian cell line.

Avipoxviruses have many advantages and are being increasingly employed as recombinant vaccine vectors. One attractive feature is that while inserted transgenes are expressed in immunologically favourable ways, avipoxvirus infections of mammalian cells are believed to be abortive. The experimental evidence supporting this belief is, however, based on a limited number of mammalian cell-types and a few avipoxvirus species. We evaluated two avian and eight mammalian cell lines for permissivity to three avipoxvirus strains, one reference fowlpoxvirus and two newly isolated strains from sparrow and pigeon, respectively. Both avian cell lines were, as expected, permissive for all three avipoxvirus strains. However, by multiplication assays, we found to our surprise that Syrian baby hamster kidney (BHK-21) cells were equally permissive to all virus strains. Results from electron microscopy of infected BHK-21 cells revealed viral morphogenesis proceeding to various forms of infectious viruses. These results were supported by the demonstration of avipoxvirus specific late gene expression and avipoxvirus specific DNA restriction pattern in BHK-21 infected cells.

Characterization of avipoxviruses from wild birds in Norway.

Avipoxviruses from different geographic regions of the world have been characterized to study their genetic and biological properties, but so far, no such work has been performed on Norwegian isolates. Lesions suggestive of avian pox, found on a Norwegian wild sparrow (Passer domesticus) and wood pigeon (Palumbus palumbus), were obtained in 1972 and 1996, respectively. Histologically, these lesions were demonstrated to be characteristic of poxvirus infections and the poxvirus was observed using an electron microscope. The resulting viruses were propagated in chicken embryo fibroblast cells. Restriction fragment length polymorphism of genomes from 2 Norwegian isolates and fowl pox vaccine strain, generated by BamHI, revealed a high degree of heterogeneity among the isolates. The profiles of avipoxviruses isolated from wild birds were clearly distinct from each other and also to the fowl poxvirus strain. Furthermore, chickens experimentally infected with pigeon poxvirus had higher antibody titers and extensive lesions compared to other isolates. This may suggest that pigeon poxvirus is more virulent than the other isolates.

Transcriptional response of immune genes in gills and the interbranchial lymphoid tissue of Atlantic salmon challenged with infectious salmon anaemia virus.

Previously, it has been assumed that fish lack organized mucosa-associated lymphoid structures. Recently, an interbranchial lymphoid tissue (ILT) was described in salmonid gills at a site with substantial exposure to antigen. In this study, immune responses were examined in gills, mid-kidney and the laser-dissected ILT of Atlantic salmon (Salmo salar L.) infected with infectious salmon anaemia virus (ISAV). A strong innate response was observed in gills and mid-kidney and even in the laser-dissected ILT, despite the fact that no virus could be traced in this tissue. A small delayed increase in IgT transcripts, exclusively in the ILT, could indicate that this tissue has a role as a secondary lymphoid organ with clonal expansion of IgT expressing B-cells. Compared to the other examined tissues, gills displayed the earliest replication of the virus, further supporting this tissue as the main entry route for infection with ISAV.

Morphogenesis of fowlpox virus in a baby hamster kidney cell line.

Fowlpox virus (FWPV) recombinant vaccines are presently being tested as an antihuman immunodeficiency virus vaccine for humans. However, biosafety, as well as the morphogenesis of FWPV in mammalian cells, are not well understood. Currently, electron microscopy is the method of choice for analyzing virus morphogenesis in cell lines. In this study, four different electron microscopic techniques were used to study FWPV morphogenesis in the Syrian baby hamster kidney (BHK-21) cell line: direct negative stain electron microscopy, ultrathin section transmission electron microscopy, cryoimmunoelectron microscopy, and scanning electron microscopy. The study showed matured viruses, as well as other stages of fowlpox virus maturation, in BHK-21 cells that led to productive virus multiplication. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. In addition, intracellular mature virus was observed in layers of the trans-Golgi network, a characteristic of intracellular mature virus wrapping that results in the formation of intracellular enveloped virus. The size and morphology of FWPV observed in this study are comparable with previously published data. This study presents the first morphological evidence for the release of FWPV by budding in BHK-21 cells.