INFECTIOUS SALMON ANEMIA

 

The first outbreaks of infectious salmon anemia (ISA) was reported among salmon parr in a hatchery on the South West Coast of Norway in 1984. Since 1985 ISA has caused massive economic losses in the Atlantic salmon (Salmo salar L.) farming industry in Norway. No outbreaks of ISA have been reported outside Norway until recently, when the virus was observed both in fish farms on the East Coast of Canada (Lovely et al., 1999) and in Scotland. It is not known whether the ISAV is present in wild salmonids. In transmission experiments, ISAV has been found to replicate both in brown trout (salmo trutta L.) and in rainbow trout (Onchorhynchus mykiss Walbaum) without causing disease (Nylund et al., 1995; Nylund and Jakobsen, 1995; Nylund et al., 1997). Furthermore, it has been suggested that ISAV may be transmitted by sea lice (Caligus elongatus and Lepeophtheirus salmonis) (Nylund, Wallace, and Hovland, 1993), although it is not known weather ISAV replicates in the lice. The pathological findings in Atlantic salmon with ISA include severe anemia, leucopenia, ascites, haemorrhagic liver necrosis and congestion of the liver, spleen and foregut (Evensen, Thorud, and Olsen, 1991; Thorud, 1991; Thorud and Djupvik, 1988). In 1994 a long term cell line, developed from Atlantic salmon head kidney macrophages (SHK-1), that supports the growth of ISAV was established (Dannevig, Falk, and Namork, 1995). Many properties of ISAV have been revealed. The virus is enveloped, as suggested first by its ether and chloroform instability (Thorud, 1991) and later by electron microscopy of the virus budding from endothelial cells (Hovland et al., 1994). Furthermore, ISAV is slightly pleomorphic, with a diameter of 100 to 130 nm in diameter containing granules of 10 to 12 nm (Hovland et al., 1994), and 10 nm surface projections (Dannevig, Falk, and Namork, 1995). Moreover, detection of ISAV antigen in the nuclei of infected cells (Falk and Dannevig, 1995) suggests that the nucleus is involved in virus replication. Evidence that ISAV induces virus-cell fusion at an acidic pH (Dannevig, Falk, and Press, 1995) indicates that infection of SHK-1 cells by ISAV depends on a low-pH step, i.e., that ISAV utilizes the endocytic pathway to infect cells. Furthermore, it has been shown that ISAV hemagglutinates piscine, but not mammalian or avian erythrocytes (Falk et al., 1997). However, spontaneous elution and the lack of reagglutination of hemagglutinated piscine erythrocytes suggest the presence of receptor-destroying enzyme activity. ISAV possesses an acetylesterase activity, but no neuraminidase activity has been detected (Falk et al., 1997). Genomic characterization has shown that ISAV has a single stranded RNA genome that consists of eight segments with a tentative negative polarity (Mjaaland et al., 1997). Taken together, the data demonstrate that ISAV shares many of the properties typical for members of the Orthomyxoviridae family.

The polymerase protein (PB1) from ISAV has recently been sequenced and found to share significant homology with other viruses, and phylogenetic studies using this sequence group the ISAV with other members of the orthomyxoviridae. Distance calculations based on the polymerase sequences indicate that the ISAV is distantly related to other members of the family. It has been proposed that ISAV may represent an old group of aquatic orthomyxoviruses based on the genetic distance calculations and a new genus is suggested to be named Aquaorthomyxoviridae to reflect the host range of ISAV as well as the proposed family allocation (Krossøy et al., 1999).

 

Adsorption and entry of orthomyxovirus into cells

Influenza viruses bind to sialic acid residues by means of the receptor-binding site in the distal tip of the HA molecule. Different influenza viruses have different specificities for sialic acid linked to galactose by a2,6- or a2,3-linkages, and this is dependent on specific residues in the HA receptor-binding pocket (Weis et al., 1990). Although the interaction of HA with sialic acid is of fairly low affinity, a high avidity of the virus for cell surfaces is probably achieved by multiple low-affinity interactions. Influenza viruses enter the cells by receptor-mediated endocytosis. The viruses are internalized in clathrin-coated membrane bound vesicles formed by invagination of specialized coated-pit domains of the plasma membrane. Following internalization the clathrin coat is removed and vesicles fuse with early endosomes. The early endosomes mature to late, more acidic, endosomes. The acidification of endosomes is brought about by H+-ATPases. The uncoating of influenza virus is dependent on the acidic pH in the endosomes (Matlin et al., 1982).

For the influenza RNPs to penetrate into the cytosol, they have to cross the membrane of the virion and that of endosomes. This is accomplished by HA-mediated fusion of the viral membrane with the cellular membrane (Fig. 1). The precise time and location of penetration depends on transition of HA to its low-pH conformation (Stegmann et al., 1987).

It is generally believed that once a virion particle has been endocytosed, the low pH activated ion channel activity of the virion-associated M2 protein (Holsinger et al., 1994; Pinto, Holsinger, and Lamb, 1992; Wang et al., 1993) permits the flow of ions from the endosome to the virion interior to disrupt protein-protein interactions and to free the RNPs from the M1 protein.

 

Figure 1

Schematic diagram of the life cycle of influenza virus (Lamb and Krug, 1996). See text for details of the model.

 

Sialic acid as receptor for Orthomyxovirus

The interaction of influenza viruses with the sialic acid on the surface of target cells is affected by several factors, such as type of sialic acid (Fig. 2), type of linkage, structure of the oligosaccharide, and the spatial arrangement of the sialic acid residues. Because of these restrictions not every sialoglycoconjugate is a suitable receptor. The important role of sialic acid in the interaction between influenza viruses and cells became evident for the first time when the receptor-destroying enzyme of influenza A and B viruses was shown to release N-acetylneuraminic acid from soluble hemagglutinin inhibitors (Fig. 4) (Klenk, 1955). The receptor-binding protein on the viral surface, the hemagglutinin, and the receptor determinant on the cell surface, have been analyzed in detail. Sialic acid is present on many glycoproteins and glycolipids, and many cells may contain a large number of potential receptors making it difficult to assign a receptor function to an individual protein. There are, however, several restrictions that limit the number of sialic acids that serves as binding sites for influenza viruses. Influenza C virus, for example, only attaches to receptors that contain N-acetyl-9-O-acetylneuraminic acid (Fig. 3) (Herrler and Klenk, 1987; Rogers et al., 1986). This type of sialic acid is not recognized by influenza A and B viruses, which have a preference for N-acetylneuraminic acid. Another restriction is the linkage type. Human influenza A viruses usually recognize sialic acid attached to galactose in an a2,6-linkage, while the avian and equine strains of the same hemagglutinin subtype bind to a2,3-linked sialic acid (Rogers and Paulson, 1983). Virus binding may also be affected by the linkage of the oligosaccaharide to peptides or lipids, for instance, whether the sialic acid is present on an aspargine-linked N-glycan or on a serine/threonine-linked O-glycan. Binding of a single sialic acid residue to the hemagglutinin of influenza A virus has been shown to be comparatively weak (Takemoto, Skehel, and Wiley, 1996). An efficient binding of virions requires a multivalent interaction between virus and cell surface receptors. Therefore, the degree of sialylation and the spatial arrangement of the sialic acids on the cell surface are also important for the attachment of influenza virions to cells. Because of these considerations, the actual number of potential receptors for influenza viruses is expected to bee much lower than the total number of surface sialoglycoconjugates.

 

 

Figure 2

Structure of major naturally occurring molecular species of sialic acids, Neu5Ac, Neu5Gc and Neu5,9Ac (Suzuki, 1994).

 

Figure 3

Structure of N-acetyl-9-O-acetylneuraminic acid, the receptor for influenza C virus. The sialic acid is shown attached to galactose in the two most common linkage types. The a2,3-linkage (top) and the a2,6-linkage (bottom) (Zimmer, Klenk, and Herrler, 1996).

 

Receptor-destroying enzymes

 

The neuraminidase integral membrane protein in influenza A and B is the second subtype specific glycoprotein of the influenza virion. It is a homotetramer of 220 kDa that can be released from the virion surface by protease treatment. It catalyzes the cleavage of the a-ketosidic linkage between terminal sialic acid and an adjacent D-galactose or D-galactosamine (Figure 4) (Gottschalk, 1957). In influenza C, the receptor-destroying enzyme of influenza C is a neuraminate-9-acetyl esterase (Herrler et al., 1987) (Figure 4), and it is a part of the HEF protein. In ISAV, a similar enzyme activity have been reported by Falk and coworkers (Falk et al., 1997). The role of this enzyme activity in the viral cycle is still unclear, but viral particles without this activity forms aggregates on the surface of infected cells (trapped like insects on a fly paper), indicating that removal of receptors is one function (to facilitate spread to neighbouring cells). This enzyme may also facilitate transport of the virus through the mucin layer of the respiratory tract . As the amino acids lining the active site of neuraminidase are invariant from strain to strain, development of  specific inhibitors of this enzyme have been a very active field of research the last years. At present, two promising inhibitors are in clinical trials (Fleming, 1999; Tai et al., 1998).

 

 

Figure 4

Cleaving sites for the receptor destroying enzyme of influenza A, B and C viruses (Suzuki, 1994).

Orthomyxovirus host range

Influenza A viruses infect swine, horses, seals, and a large variety of birds as well as humans (Easterday, 1975). Influenza B viruses on the other hand, infect only humans (Baine, Luby, and Martin, 1980). Influenza C viruses infect human and swine. Phylogenetic studies of influenza A viruses have revealed species-specific lineages of viral genes and have demonstrated that the prevalence of interspecies transmission depends on the animal species. It has also been revealed that aquatic birds are the source of all influenza viruses in other species (Hinshaw et al., 1981).

ISAV have also proven to be able to infect a range of  fish species. In transmission experiments, ISAV has been found to replicate both in brown trout (salmo trutta L.) and in rainbow trout (Onchorhynchus mykiss Walbaum) without causing disease (Nylund et al., 1995; Nylund and Jakobsen, 1995; Nylund et al., 1997). Furthermore, it has been suggested that ISAV may be transmitted by sea lice (Caligus elongatus and Lepeophtheirus salmonis) (Nylund, Wallace, andHovland, 1993), although it is not known weather ISAV replicates in the lice.

 

INFECTIOUS PANCREATIC NECROSIS VIRUS

 

IPNV is a member of the virus family Birnaviridae and causes an acute contagious disease in a number of economically important fish species. The disease is characterized by necrosis of pancreatic cells and high mortality (Dobos et al., 1979). The virions are nonenveloped, 60 nm icosahedral particles with the outer surface composed of VP2,  the capsid glycoprotein. The two segment (3 kb and 2.8 kb) doublestranded RNA genome also encodes four additional viral proteins (VP1, preVP2, VP2, VP3 and VPg)(Hsu, Chen, and Wu, 1995). Infected cells also contain NS or VP4, a virus encoded protease and a positively charged minor protein. The biology of this virus has been extensively at a molecular level and all genes are cloned (Dobos and Roberts, 1983). Although there are some studies on the mode of attachment and entry into cells, no cellular receptor has yet been identified (Couve, Kiss, and Kuznar, 1992; Farias et al., 1988; Kuznar et al., 1995). This part of IPN biology therefore seems to be well suited for our types of studies.

 

Problems

 

In the last 2.5 years we have investigated binding and uptake of ISAV in SHK-1 cells. Our results shows that the binding to both cells and electroblotted membrane-proteins from SHK-cells is neuraminidase sensitive. Binding can be increased at low pH and abolished by periodate treatment (oxidation of sialic acid residues). We have also shown by confocal fluorescence- and electron microscopy that entry of virus takes place from the endosomes/lysosomes. Furthermore, we could show fusion of virion particles with the plasma membrane upon lowering the pH in the medium. These results have been published in Journal of Virology (Eliassen et al., 2000).

            The activity of the receptor destroying enzyme have also been in the focus of our research. We are in the process of analyzing enzyme kinetics and establishing an inhibitor profile using known acetyl esterase inhibitors. There are at least 3 types of acetyl esterases (A,B and C) were influenza C esterase belongs to a group of serine esterases  that can be inhibited by diisoflourophosphate (DFP).  We are now in the process of  classifying the ISAV esterase using a range of esterase inhibitors (manuscript 3).

            The main focus of our further research will be on identifying on a molecular level the type of sialic acid recognized by the ISAV HA molecule. This will be done by a virus overlay method using  sialoconjugates separated on thin layer chromatography. When chromatograms are overlayed with radioactive or fluorescent virus particles, the sugars can be identified. We have already used this methods successfully on protein blots. The next important question to be adressed is whether the HA activity and esterase activity resides on the same protein molecule, as is the case for the influenza C HEF molecule. This will be done by 2-D SDS-PAGE and blotting experiments. The HA molecule can be identified by the neutralizing antibody developed by Falk (Falk, Namork, and Dannevig, 1998), whereas the esterase can be identifiied by a chromogenic substrate (Fast red) method. We are already on the way with these experiments.

            Our ultimate goal is to clone the gene or genes coding for these proteins. We have established methods for purification of virus and isolation of viral RNA. We have also contructed  a cDNA library that contains (controlled by PCR) the already cloned segments from ISAV (Krossøy et al., 1999; Mjaaland et al., 1997). Hopefully we will be able to contruct expression libraries that contain the antigen / enzyme activity of interest.

            These projects will be conducted I cooperation with several laboratories at a national and international level. Together with researchers at the vetrinary institute I heve sent grant applications for these projects both to NFR and EU.

 


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