key: cord-0787013-e407g5ha authors: Kibenge, F.S.B.; Kibenge, M.J.T. title: Orthomyxoviruses of Fish date: 2016-09-09 journal: Aquaculture Virology DOI: 10.1016/b978-0-12-801573-5.00019-x sha: e44421730479e53b98ef6b0ada4487959df17622 doc_id: 787013 cord_uid: e407g5ha The family Orthomyxoviridae is well known for containing influenza viruses with a segmented RNA genome that is prone to gene reassortment in mixed infections (known as antigenic shift) resulting in new virus subtypes that cause pandemics, and cumulative mutations (known as antigenic drift), resulting in new virus strains that cause epidemics. This family also contains infectious salmon anemia virus (ISAV) and tilapia lake virus (TiLV), which are a unique orthomyxoviruses that infect fish and is unable to replicate above room temperature (24°C). This chapter describes the comparative virology of members in the family Orthomyxoviridae in general, helping to understand the emergent teleost orthomyxoviruses, ISAV and TiLV. The most current information on virus–host interactions of the fish orthomyxoviruses, particularly ISAV, as they relate to variations in virus structure, virulence, persistence, host range and immunological aspects is presented in detail. . b A new genus approved in 2013 (Presti et al., 2009; Adams, M.J., King, A.M.Q., Carstens, E.B., 2013 . Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2013) . Arch. Virol. 158, 2023 -2030 . c Tilapia lake virus (TiLV) is a novel orthomyxo-like virus causing mass die-offs of tilapia in Israel and Ecuador, representing a new unnamed genus in the family Orthomyxoviridae (Bacharach et al., 2016) . Gene products: PB2, polymerase basic protein 2; PB1, polymerase basic protein 1, **(a second protein PB1-F2 varies in length from 11 to 90 amino acids; a third protein PB1 N40 is 718 amino acids long in some strains Influenzavirus A); PA, polymerase acidic protein (P3 in Influenzavirus C and D as it lacks the acidic features at neutral pH (Yamshita, M., Krystal, M., Palese, P., 1989 . Comparison of the three large polymerase proteins of influenza A, B, and C viruses. Virology 171, [458] [459] [460] [461] [462] [463] [464] [465] [466] ); NP, nucleoprotein; HA, hemagglutinin in two subunits in Influenzavirus A and B; HEF, hemagglutinin-esterase-fusion in Influenzavirus C (Herrler, G., Durkop, I., Becht, H., Klenk, H.-D., 1988 . The GP of influenza C virus is the hemmagglutinin, esterase, and fusion factor. J. Gen. Virol. 69, [839] [840] [841] [842] [843] [844] [845] [846] and Influenzavirus D (Hause et al., 2014) ; F, fusion protein in Isavirus (Aspehaug et al., 2005) ; HE, hemagglutinin-esterase in Isavirus ; NA, neuraminidase; M1, matrix protein 1; M2, matrix protein 2/ion channel protein; NS1, nonstructural protein 1; NS2, nonstructural protein 2 (is also known as nuclear exporting protein, NEP, and is present in purified viral preparations); NS3, nonstructural protein 3 in Isavirus (Kibenge et al., 2007a,b) . c Influenzavirus D is a new genus proposed in 2014 (Hause et al., 2014) . Clouthier et al., 2002). b Values obtained from cDNA of genomic segments of six genetically distinct ISAV isolates: NBISA01 and ADL-ISAV-07 belonging to two different genotypes of ISAV (Kulshreshtha et al., 2010), 390/98 Scotland, 982/08 Scotland, Glaesver 2/90 Norway and RPC/NB 98-049 Canada (Fourrier et al., 2011) , and Glaesver 2/90 Norway (Mérour et al., 2011). The 5' and 3' untranslated regions (UTRs) are written for the positive sense (cRNA), and are equivalent to the respective 3' and 5' noncoding regions (NCRs) of the negative sense RNA segments (vRNA). The total length includes the range between the shortest and longest 5' and 3' UTRs for each segment among the six ISAV isolates (Kulshreshtha et al., 2010; Fourrier et al., 2011) . The 3' UTR does not include the stop codon. Numbers in brackets denote # nucleotides to polyadenylation signal, and length of polyadenylation signal. c Protein nomenclature and data used by Cottet et al. (2011) and Mérour et al. (2011) for ISAV. See also Cottet et al. (2011) for ISAV and from Bouvier and Palese (2008) for influenza A virus. e A novel orthomyxolike virus Tilapia lake virus (TiLV), with 10 genome segments with no recognizable homology to other known sequences, except for segment 1, which predicts a protein with weak homology to the PB1 subunit of Influenzavirus C, was recently described (Bacharach et al., 2016) . A complete sequence of the PB1 gene of a putative koi carp orthomyxovirus was obtained from koi carp in California with 43% amino acid sequence identity with ISAV (Anonymous, 2013) . The clinical disease, infectious salmon anemia (ISA), in farmed Atlantic salmon (Salmo salar) was first recognized in Atlantic salmon in 1984 on the southwestern coast of Norway (Thorud and Djupvik, 1988) , although the virus might have been present in Norwegian salmon farms as early as 1977 or 1978 (Nylund et al., 1995a; Devold et al., 2001) . The situation developed into an epidemic, which peaked in 1990, forcing the Norwegian authorities to impose a series of biosecurity measures that significantly reduced but not eliminated disease outbreaks (Håstein et al., 1999) . The World Organization for Animal Health (OIE) recognized the viral disease in 1990 and named it ISA, and it has been an OIE-notifiable disease ever since. The causative agent, ISAV, was first detected by electron microscopy and virus isolation from tissues of infected fish in 1995 (Dannevig et al., 1995a,b) , which allowed its molecular characterization and subsequent classification to the family Orthomyxoviridae ), genus Isavirus (McCauley et al., 2012 . The disease was first reported outside Norway in 1996, in New Brunswick, Canada (Byrne et al., 1998; Mullins et al., 1998) , although there is anecdotal evidence indicating that the virus was present in the Bay of Fundy by 1995. Subsequently, ISA outbreaks were reported in Scotland, in the Faroe Islands, Denmark, in Maine, and in Chile in 2007 (reviewed by Cottet et al., 2011) . The economic impact of the disease was probably most apparent in Chile, where losses during the 2007-11 ISA outbreaks were estimated at about $1 billion (ie, 50% of the economic value of the industry) (Alvial et al., 2014) . More recently, ISA outbreaks have occurred in Nova Scotia and Newfoundland, Canada (CFIA, 2014) . ISAV is now endemic in fish populations in Norway, New Brunswick, Canada, Scotland, Faroe Islands, Maine and Chile, and virulent strains of the virus have been replaced by low-pathogenic variant ISAVs called ISAV-HPR0 viruses. The field disease has only been found in farmed Atlantic salmon and is characterized by high mortality with exophthalmia, pale gills, ascites and hemorrhagic liver necrosis, renal interstitial hemorrhage and tubular nephrosis. No natural outbreaks of ISA have been reported in fish other than farmed Atlantic salmon, although the virus can be detected by reverse transcription polymerase chain reaction (RT-PCR) in feral fish Plarre et al., 2005; EFSA, 2012) . Laboratory data on tissues and sera from farmed coho salmon (Oncorhynchus kisutch) indicated the existence of ISAV in Chile as early as 2000 (Kibenge et al., 2001a (Kibenge et al., , 2002 with some fish showing jaundice, although no ISA outbreak was detected in neighboring Atlantic salmon farms (Smith et al., 2006) . The virus has also been detected in apparently healthy farmed rainbow trout (Oncorhynchus mykiss) in Ireland in 2002 (Anonymous, 2002) . ISAV is an economically important pathogen in marine aquaculture. It is the only species of the genus Isavirus, one of the seven genera of the family Orthomyxoviridae (Table 19 .1; Fig. 19 .2). However, a complete sequence of PB1 gene of a putative koi carp orthomyxovirus was obtained from koi carp in California with 43% amino acid sequence identity with ISAV (Anonymous, 2013) , and there is also an independent reference to an orthomyxovirus from koi carp (Granzow et al., 2014) . Most recently, the complete genome sequence of a novel orthomyxo-like virus Tilapia lake virus (TiLV) with 10 genome segments was reported (Bacharach et al., 2016) , and likely belongs to a new unnamed eighth genus in Orthomyxoviridae. Viruses in the genus Isavirus are enveloped particles with a diameter of 90-140 nm (Dannevig et al., 1995a,b; Nylund et al., 1995a,b) , with 13-15-nm-long, mushroom-shaped surface projections consisting of a combined receptor-binding HA and receptor-destroying enzyme activity demonstrated to be an esterase, designated as HE Hellebø et al., 2004) and fusion GP (Aspehaug et al., 2005) , designated as F. This arrangement of the functional activities of the surface proteins in ISAV is in contrast to other orthomyxoviruses (which have hemagglutinating and fusion activities on one virus protein and the receptor-destroying enzyme activity on a separate virus protein), but is similar to most paramyxoviruses (reviewed by Aamelfot et al., 2014) . The genome is composed of eight segments of linear, single-stranded negative sense RNA ranging in length from 1.0 to 2.4 kb (see Table 19 .3 above) with a total molecular size of approximately 14.3 kb (Clouthier et al., 2002) . The buoyant density of the complete virus particles in sucrose and cesium chloride gradients is 1.18 g/mL . All eight RNA segments of ISAV have been sequenced Krossøy et al., 1999 Krossøy et al., , 2001 Rimstad et al., 2001; Ritchie et al., 2001 Ritchie et al., , 2002 Cunningham and Snow, 2000; Clouthier et al., 2002; Snow et al., 2003) . Comparison of the ISAV proteins with those of other orthomyxoviruses revealed low amino acid identity values, between <13% and <25% Kibenge et al., 2001b; Snow and Cunningham 2001; Ritchie et al., 2002) . The most conserved proteins between ISAV and influenza virus A/PR/8/34 are PB1 proteins, although they share only 31% amino acid similarity (Mérour et al., 2011) . Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins (Ritchie et al., 2002; Lamb et al., 1981) . In a subsequent study, Kibenge et al. (2007a) showed that ISAV segment 7 encodes three proteins with estimated molecular masses of 32, 18 and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on the removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively ( Fig. 19 .3). The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids (Kibenge et al., 2007a) . The functions of the orthomyxovirus proteins are summarized in Table 19 .3, using genus Isavirus as an example. Genetic reassortment in ISAV has been indicated by molecular and phylogenetic sequence analyses (Cottet et al., 2010; Devold et al., 2006; Markussen et al., 2008) . The F protein of some ISAV strains contains insertions near the putative proteolytic cleavage site, acquired through nonhomologous recombination (Devold et al., 2006; Markussen et al., 2008; Kibenge et al., 2009) . The virulent strains of ISAV have deletions in a highly polymorphic region (HPR) in the stem of the HE protein (designated as ISAV-HPRΔ). The low pathogenic or nonvirulent strains of ISAV (designated as ISAV-HPR0) have a full-length sequence (35 amino acids) of HPR and are considered to have an ancestral relationship with ISAV-HPRΔ (EFSA, 2012). The deletions in HPR probably occur through homologous recombination (copy-choice recombination, presumably because of strand-switching by the vRNA polymerase (Castro-Nallar et al., 2011) during negative RNA strand synthesis from one nucleic acid template of one virus to another (which has been shown to occur in picornaviruses, coronaviruses, influenza virus, alphavirus, rotavirus and orbivirus). The novel ISAV strains (ISAV-HPRΔ) were suggested to be produced during intensive aquacultural practices (Mjaaland et al., 2005) . RNA molecules of members of the family Orthomyxoviridae possess conserved and partially complementary 5'-and 3'-end sequences characteristic of each genus. These conserved terminal nucleotide sequences are therefore useful for orthomyxovirus classification. In Influenzavirus A, the first 12 nucleotides at the 3' end and the first 13 nucleotides at the 5' end of noncoding regions (NCRs) in all the vRNA segments are highly conserved (Skehel and Hay, 1978; Robertson, 1979; Desselberger et al., 1980; de Wit et al., 2007; Bouvier and Palese, 2008; Wang and Lee, 2009 ). These partially complementary termini base pair form terminal panhandle structures (Hsu et al., 1987) , which function as promoters by interacting with the viral polymerase complex during replication and transcription of vRNA (de Wit et al., 2007; Li and Palese, 1994; Hagen et al., 1994; Lee et al., 2003; Fodor et al., 1995; Luyjtes et al., 1989; Tchatalbachev et al., 2001) . Moreover, the segmentspecific NCR sequences may play important roles in virus virulence (Zheng et al., 1996) and in the rescue of influenza virus using the reverse genetics system (de Wit et al., 2007; Jackson et al., 2002) . The sequences for the genomic ends of ISAV RNA have been reported only recently (Kulshreshtha et al., 2010; Mérour et al., 2011; Fourrier et al., 2011) . To assist with comparison between different reports, we have adopted the following terminology: 5' and 3' untranslated regions (UTRs) when referring to the positive sense (cRNA), which correspond to 3' and 5' NCRs of the negative sense RNA segments FIGURE 19.3 New gene expression model for segment 7 of ISAV of North American genotype. There are three ORFs, consisting of the primary transcript (7-ORF1) which is 300 amino acids long, and 7-ORF1/2 and 7ORF1/3, each with an intron removed from 7-ORF1 and consisting of 159 and 81 amino acids, respectively. From Kibenge, F.S.B., Xu, H., Kibenge, M.J.T., Qian, B., Joseph, T., 2007a . Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus. Virol. J. 4, 34. (vRNA). The length of the 5' NCR in all eight ISAV segments is significantly longer than the corresponding 3' NCR. It is also variable in the different genome segments of the same ISAV strain and on the same genome segment in different ISAV strains, ranging from 67 nucleotides in segment 4 to 150 nucleotides in segment 3 (Table 19 .3). Moreover, Kulshreshtha et al. (2010) was able to document the presence of intrasegment ISAV quasispecies based on sequence variation in the NCR sequences of the transcripts. However, the 3' terminal 7 nucleotides and the 5' terminal 11 nucleotides are highly conserved among the eight genomic segments in the different ISAV strains (3′-UCG U / A UUC--------GU U / A AAAA A / U UGA-5') ( Fig. 19.4 ). In the case of TiLV, the conserved NCR sequences of its 10 genome segments (Bacharach et al., 2016; see Fig. 19 .7) differ from those of ISAV, which is consistent with both viruses from different genera. Of note is the strict conservation of the terminal 3 and 4 nucleotides at the 3' and 5' ends, respectively, of vRNA in all orthomyxoviruses, which is indicative of a shared mode of replication requiring interaction of the viral ends with the polymerase complex (Fourrier et al., 2011) . Toro-Ascuy et al. (2015) reported for the first time the successful establishment of a plasmid-based reverse genetics system for the rescue of ISAV. The system corresponds to the 8+4 plasmid system for the reverse-genetics system for influenza A virus (Neumann et al., 1999) . Thus, the full-length viral cDNA clones (having the entire coding and noncoding regions) of all eight ISAV RNA genomic segments were individually constructed into pUC57 under the control of the Atlantic salmon internal transcribed spacer region 1 (ITS-1) as a promoter flanked by the sequences of the hammerhead ribozyme in the 5' end and the hepatitis δ virus ribozyme in the 3' end, followed by the transcription terminator of the rabbit β-globin in pSS-URG (for plasmid for Salmo salar universal reverse genetics) plasmid, creating eight genomic plasmids. In addition, four plasmids expressing the four proteins (PB2, PB1, PA and NP) of the ISAV ribonucleoprotein complex under the control of the cytomegalovirus (CMV) promoter were constructed. Transfection of ASK cells with the 12 plasmids resulted in the rescue of infectious recombinant ISAV (rISAV) 7 days posttransfection (Toro-Ascuy et al., 2015) . The concept of ISAV strain variation has gained wide acceptance since Blake et al. (1999) and Inglis et al. (2000) first reported partial genomic sequence data of segments 2 and 8, showing significant differences between Canadian and Norwegian isolates . It is now well established that sequence analysis of all eight genomic segments from different ISAV isolates consistently reveals two genotypes designated according to their geographic origin as European (Genotype I) and North American (Genotype II) (Godoy et al., 2008) . Genotype I can be further subdivided into Genogroup 1 (European-in-North America, EU-in-NA) for Genotype I strains also found in North America, and Genogroup 2 (Real European, Real-EU) for Genotype I strains only found in Europe and Chile . The ISAV of the European genotype can also be differentiated into three genogroups, EU-G1, EU-G2 and EU-G3; the EU-in-NA strains are placed in the EU-G2 group, and Real-EU strains are divided between the EU-G1 and EU-G3 groups (Nylund et al., 2007) . Because the two ISAV genotypes correlated to antigenic groupings, they were designated as HA subtypes (Kibenge et al., 2001b) . The two HA subtypes of ISAV have nucleotide sequence identities of ≤80.4% and amino acid sequence identities of ≤88.2%, whereas within each subtype, both sequence identities are ≥90.7% (Marshall, 2003) . For influenza A virus, which has up to 18 different HA subtypes, amino acid sequence identities for isolates belonging to different subtypes can range from 25% to 80% (Nobusawa et al., 1991; Kawaoka, 1991; Wright et al., 1995; Shekel and Wiley, 2000) . Thus, the HA protein of ISAV shows less variation than that of the influenza A virus. Limited virus neutralization tests with rabbit antisera to ISAV isolates of Real-EU genogroup (Genotype I, Genogroup 2) and North American genotype (Genotype II) showed at least three HA subtypes of ISAV: (1) North American, (2) Real-EU and (3) EU-in-NA . Alignment of deduced amino acid sequences in the HE protein of selected ISAV isolates identified the putative antigenic motif around the common potential N-glycosylation site at amino acid positions 333 NIT 335 with one mutation site at positions 320 to 323 on one side and a second mutation site at positions 339 to 367, between European and North American genotypes. The HE protein of ISAV isolates of EU-in-NA genogroup has an additional unique potential N-glycosylation site at amino acid positions 362 NQT 364 (Marshall, 2003) . The presence of the additional carbohydrate chain in this region may be the basis for a new epitope accounting for EU-in-NA genogroup being antigenically distinct from the Real-EU genogroup . Strain designation of ISAV is currently based on sequence deletions in the 35-amino acid HPR of the HE protein encoded on segment 6. The virulent strains of ISAV, designated ISAV-HPRΔ, have deletions in HPR of the HE protein and are named numerically from HPR1 to ≥HPR30, with two of them (HPR20 and HPR21) of North American genotype and the rest of them of European genotype (Nylund et al., 2007; Kibenge et al., 2009 ). The low pathogenic or nonvirulent strains of ISAV designated ISAV-HPR0, have a full-length sequence (35 amino acids) of HPR and are considered to have an ancestral relationship with ISAV-HPRΔ (EFSA, 2012). Using multiple alignment and phylogenetic analysis of segment 6 sequences from all ISAV-HPR0 viruses reported worldwide, Godoy et al. (2013) identified three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 ( 360 PST 362 , 360 PAN 362 and 360 PAT 362 ) in HPR. The ISAV-HPR0 residue pattern 360 PAT 362 is the only type of ISAV-HPR0 found in Chile. The pathogenic ISAV isolates also have a mutation at the proteolytic cleavage site ( 267 R) of the F protein Kibenge et al., 2009 ). This mutation, relative to the F protein of ISAV-HPR0, is either a single amino acid change from glutamine ( 226 Q) to lecine ( 226 L) or a peptide insert, named numerically from IN1 to IN5 (Godoy et al., 2014) . IN1 to IN3 have been detected in Norwegian isolates (Devold et al., 2006) and IN4 and IN5 only in Chilean isolates Godoy et al., 2014) . Thus, there is still a need to devise an ISAV strain designation system that takes into account both envelope proteins (HE and F), which have been successfully used in genotyping of ISAV . Godoy et al. (2014) , used concatenated ISAV F and HE genes of one representative isolate from every geographical region where ISA has been reported (ISAV isolates chosen: 98-049-1 represents North America; 04-085-1 represents EU-in-NA; 810/9/99 represents Norway I; 93/09/2264 represents Norway_II_1; SK779/06 represents Norway_II_2; U24636 represents Chile; and 390/98 represents Scotland) and constructed a phylogenetic tree that revealed the relationship between the different geographical isolates and possible transmission routes among the regions. ISAV isolates could also be grouped into three phenotypes based on their ability to grow and cause cytopathic effects (CPEs) in CHSE-214 cells , but the molecular basis for this phenotypic variation has not yet been determined. The different ISAV isolates also vary in the molecular sizes of their polypeptides (Kibenge et al., 2000; Griffiths et al., 2001) , as do influenza viruses (Kendal, 1975) . The replication strategy of ISAV resembles that of influenza viruses . The influenza viruses attach via their HA protein to host-cell receptors terminating in sialic acids (Zambon, 2001) . Hemagglutination assays with erythrocytes from several fish species have indicated that ISAV also uses its HA protein to bind sialic acid receptors on the cell surface Eliassen et al., 2000; Kristiansen et al., 2002; Workenhe et al., 2007) . Structurally diverse sialoglycoproteins, as well as certain unique sialoglycoproteins, have been reported to occur in salmonid species (Iwasaki et al., 1990) . Because ISAV agglutinates horse erythrocytes, and since these contain only N-glycol neuraminic acid, it has been suggested that this may be the cellular receptor for ISAV (Kristiansen et al., 2002) . It was subsequently demonstrated that 4-O-acetylated sialic acid (Neu4,5Ac 2 ) serves both as a substrate for the receptor destroying enzyme, a sialate-4-O esterase, and as a receptor determinant for virus binding (Hellebø et al., 2004) ; hence, the designation of the ISAV HA protein encoded on segment 6 as a HA-esterase (HE) . The 4-O-acetylated sialic acid was recently shown to be the preferred receptor for ISAV in vivo as well (Aamelfot et al., 2012) . Eliassen et al. (2000) and Aspehaug et al. (2005) demonstrated the internalization of ISAV into the endosomes and low pH-dependent fusion of the viral envelope with the endosomal membrane, releasing the viral genome. The subsequent events in the ISAV infection cycle in a fish cell, including translocation of viral ribonucleoprotein complexes to the nucleus, transcription and replication of the viral genome in the nucleus, translation of viral transcripts in the cytoplasm, transport of the proteins to the nucleus and the surface GPs through the secretory pathway from the endoplasmic reticulum, through the Golgi apparatus to the plasma membrane, formation of the ribonucleoprotein complex in the nucleus and subsequent transportation to the budding site of the plasma membrane with the surface GPs ( Fig. 19 .5), were reviewed by Cottet et al. (2011) . ISAV replicates in cell culture without trypsin treatment. Moreover, unlike in influenza viruses, cleavage of the HA protein does not occur in ISAV . In influenza A viruses, amantadine specifically inhibits the M2 ion channel activity, and therefore viral ribonucleoprotein complexes are not released into the cytoplasm. However, amantadine did not have any effect on ISAV replication . Permissive fish cell lines for ISAV include salmon head kidney (SHK-1) cells (Dannevig et al., 1995b) , TO cells (Wergeland and Jakobsen, 2001) and Atlantic salmon kidney (ASK-2) cells (Devold et al., 2000) which are macrophagelike cell lines in which virus replicates with production of CPEs. Although all three cell lines are derived from the Atlantic salmon pronephros, they are distinctly different in their growth characteristics (media requirements, split ratio, cell composition), they display distinctly different viral-induced CPEs and also interact differently with virus-antibody mixtures during the virus neutralization test . The SHK-1 and ASK-2 cell lines are widely used for primary isolation of ISAV; however, at higher passages, they seem to lose sensitivity for ISAV, resulting in poorly defined and slow developing CPEs by some ISAV strains. Some strains of ISAV can also replicate and cause CPEs in the Chinook salmon embryo (CHSE-214) cell line Kibenge et al., 2000; Griffiths et al., 2001) while others do not replicate in this cell line at all , thereby limiting their utility in virus isolation (Kibenge et al., 2000) . Moreover, for cytopathic strains, the CPEs in CHSE-214 cells develop slowly, taking up to 17 days p.i. compared to 12 days p.i. in SHK-1 cells, and the virus yields are also lower (10 4.5 to 10 6.5 TCID 50 /mL) than on the SHK-1 cell line . TO (Wergeland and Jakobsen, 2001) and ASK-2 (Devold et al., 2000) cell lines develop more easily discernible CPEs in a shorter time (by 2-4 days p.i.) than do SHK-1 and CHSE-214 cells and give higher virus yields (as high as 10 9.1 TCID 50 /mL, Wergeland and Jakobsen, 2001; Grant and Smail, 2003) . However, they do not recover viruses from some ISAV RT-PCR-positive samples (Kibenge et al., 2001a; Rimstad and Mjaaland, 2002; Mjaaland et al., 2002) , indicating that the currently available fish cell lines are not permissive for all ISAV strains. Interestingly, Aamelfot et al. (2012) found the preferred ISAV cellular receptor only in the Atlantic salmon macrophagelike cell lines (ASK-2, SHK-1 and TO), but not in the CHSE-214 cell line. SHK-1 and ASK cell lines can be used in combination to provide an enhanced ability to detect ISAV (Rolland et al., 2005) . Virus replication also occurs in Atlantic salmon (AS) (Sanchez et al., 1993; Sommer and Mennen, 1997) and rainbow trout gill (RTgill-W1) cell lines (Bols et al., 1994; Falk et al., 1997) ; but in these cases, the virus is noncytopathic and is detected by indirect fluorescent antibody testing (IFAT). The virus yield is very poor; the maximum viral titer reported in these cells was 10 3 TCID 50 /mL . Virus replication on FH-10, BB, EPC and BF-2 cell lines has also been attempted, but these cell lines appear to be refractory to ISAV Byrne et al., 1998) ; the ISAV cellular receptor was not found in the EPC and BF-2 cell lines (Aamelfot et al., 2012) . Two general pathways known to cause CPE and eucaryotic cell death during virus infection are apoptosis and necrosis. In a study using the permissive fish cell lines SHK-1, CHSE-214 and TO, Joseph et al. (2004) observed apoptosis only in ISAV-infected SHK-1 and CHSE-214 cells. ISAV-infected TO cells did not undergo apoptosis but showed leakage of high mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis (Scaffidi et al., 2002) , suggesting that CPEs in these cells are associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of the HMGB1 protein. Infection with two different strains of ISAV showed that the induction of apoptosis correlated with the appearance of CPEs in SHK-1 cells. The ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor Z-VAD-fmk, indicating a caspase activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 specifically bound caspase-8 in vitro, suggesting that these viral proteins may have a role in the ISAV-induced apoptosis. These findings demonstrated for the first time that the mechanism of cell death during ISAV infection was dependent on the cell type, which might have implications for ISAV pathogenesis and persistence. It was recently shown that ISAV infection promotes apoptosis of SHK-1 cells through ROS/p38 MAPK/Bad signaling pathway (Olavarría et al., 2015) . Several theories have been put forward to explain the geographic and host origin, and therefore the virulence, of ISAV in farmed fish (Nylund et al., 1995b (Nylund et al., , 2003 . For example, it was suggested that ISAV may have been introduced to Norway with the importation of rainbow trout from North America . It has been suggested that there are natural reservoirs for the virus, probably in fish occurring in the coastal areas where ISA outbreaks frequently occur (Mullins et al., 1999) . The virulence of ISAV is intrinsically linked to its emergence as a pathogen in marine-farmed Atlantic salmon. Thus, it is more likely that the emergence of ISAV as a fish pathogen is a reflection of variation in virushost interactions similar to the situation with avian influenza in poultry. For example, domestic poultry (of the order Galliformes), are not the natural hosts of avian influenza viruses (Perdue et al., 1999; Suarez and Schultz, 2000) . However, people have altered the epidemiological variables of avian influenza viruses by creating new ecological niches via bird captivity and domestication, industrial agriculture, national and international commerce and nontraditional raising practices (Swayne, 2000) . In these new ecosystems, pathogenic and nonpathogenic microorganisms can be transmitted within and between avian species (or fish species, in the case of ISAV) and adapt to new host species. For both European and North American genotypes of ISAV, a direct functional relationship can be demonstrated between the length of HE protein stem, ISAV cytopathogenicity in cell culture and pathogenicity for Atlantic salmon (Kibenge et al., 2006 (Kibenge et al., , 2007b . It is now well established that the systemic disease ISA is caused by virulent ISAV strains (designated as virulent ISAV-HPRΔ) with deletions in the highly polymorphic region (HPR) spanning residues 337 V to M 372 in the stem of the HE protein; these virus strains also have either an insertion or the 266 Q→ 266 L mutation in the F gene . ISAV virulence in ISAV-HPRΔ may be attenuated by the absence of the insertion in the F gene (Godoy et al., 2014) . However, while the deletion in HPR may be a good genetic marker for differentiation (Ritchie et al., 2009) , it is not necessarily the virulence determinant for ISAV. Direct evidence for how the deletion in HPR affects virulence will be possible now that a reverse genetics system for ISAV has been developed (Toro-Ascuy et al., 2015) . Noncultivable, nonpathogenic ISAV detectable only by RT-PCR has full-length HPR sequence (35 amino acids) in the HE-encoding gene and is designated ISAV-HPR0 . ISAV-HPR0 viruses have been variously reported in the research literature (Godoy et al., 2014) . All ISAV-HPR0 isolates are characterized by the wild-type F protein with Q 266 and no sequence insertion . ISAV-HPR0 replicates mainly in Atlantic salmon gills, causing only transient subclinical infection (Christiansen et al., 2011) . This virus has been detected in apparently healthy wild and farmed Atlantic salmon in most regions with Atlantic salmon aquaculture (Godoy et al., 2013) and is considered to have ancestral relationship with ISAV-HPRΔ (EFSA, 2012). ISAV-HPR0 viruses are detected late during ISA outbreaks and persist long after the disease is contained or eradicated (Kibenge et al., 2012b McBeath et al., 2009; Christiansen et al., 2011) . Most recently, ISA cases directly linked to the presence of endemic ISAV-HPR0 have been reported (Godoy et al., 2013) , supporting the notion that under appropriate conditions, ISAV-HPR0 can mutate to ISAV-HPRΔ. The clinical features and epidemiology of ISA were reviewed by Kibenge et al. (2004) . Clinical signs of ISA in farmed Atlantic salmon include anorexia, lethargy and anemia. Mortality in a fish cage on a fish farm rises slowly and can vary from 0% to 90% (cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0% to100%). The disease course is prolonged with low daily mortality (0.05-0.1%), typically only in a few cages. Virus may be present in a fish cage for up to six months before significant mortality is noted. Stressful events such as handling of fish (eg, during sorting, treatment, splitting or moving of cages) may initiate disease outbreaks on infected farms. The dead fish have exophthalmos (bulging, bloodshot eyes), pale gills due to anemia, blood-tinged fluid in peritoneal and pericardial cavities (ascites), congestion of gut, enlargement of liver and spleen and petechial hemorrhages of the internal organs and membrane lining the abdominal cavity ( Fig. 19.6 ). Histopathology lesions include intestinal mucosal ulceration and hemorrhage, liver hemorrhage and hepatocyte necrosis, erythrophagocytosis in the spleen and kidney, renal interstitial congestion, hemorrhage and tubular necrosis and congestion of branchial lamellar and filamental vessels (Byrne et al., 1998; Mullins et al., 1998; Thorud and Djupvik 1988; Evensen et al., 1991; Godoy et al., 2008) . ISAV is known to cause overt and fatal systemic infection in farmed Atlantic salmon and asymptomatic infection in feral fish, a situation analogous to avian influenza viruses in domestic poultry and wild birds. The clinical ISA disease can also occur in wild, free-ranging Atlantic salmon, but these fish are less susceptible than the farmed Atlantic salmon either due to genetic variation in the two fishes or due to stress caused by management practices on the salmon farms (Nylund et al., 1995b) . Experimental infection of Oncorhynchus spp., Arctic char (Salvelinus alpinus) and herring (Clupea harengus) with ISAV resulted only in subclinical infection (Nylund et al., , 1995b (Nylund et al., , 2002 Nylund and Jakobsen 1995; Rolland and Nylund 1999; Devold et al., 2001) . The Pacific salmonid species, chum (Oncorhynchus keta), steelhead (Oncorhynchus mykiss), Chinnook (Oncorhynchus tsawytscha) and coho (Oncorhynchus kisutch) were also found to be resistant to experimental infection with ISAV, even at doses as high as 10 8 TCD 50 /mL that induced 98% mortality in Atlantic salmon (Rolland and Winton, 2003) . Although attempts to isolate the virus from some of these fishes have not been successful, the virus can be detected by RT-PCR Plarre et al., 2005) . Detection of ISAV by RT-PCR has also been reported in the tissues of pollock (Pollachius virens) and Atlantic cod (Gadus morhua), but only in fish collected from cages with ISA-diseased Atlantic salmon . Pollock were shown to be able to eliminate the virus within a week of experimental infection . Kibenge et al. (2006) reported that coho salmon (O. kisutch) can remain asymptomatic while being infected with strains of ISAV that are highly pathogenic to Atlantic salmon. Such benign infections in the wild fishery are considered to be the source of virulent strains that cause clinical disease in marine-farmed Atlantic salmon (Murray et al., 2002) . The only other fish species conclusively shown to develop clinical disease and die due to ISAV infection are rainbow trout (O. mykiss) and Amago trout (Oncorhynchus masou macrostomus, an indigenous fish of Japan). In the case of rainbow trout, percentage mortalities of 10-50% were obtained following intraperitoneal (i.p.) injections with highly pathogenic ISAV isolates (NBISA01 and RPC/NB 98-049-1 of North American genotype and 810/9/99 of European genotype) that caused 80-100% mortality in Atlantic salmon (Kibenge et al., 2006; MacWilliams et al., 2007) . Of the gross lesions reported for ISA in Atlantic salmon, only visceral adipose petechiation and ascites were commonly observed among the rainbow trout mortality (MacWilliams et al., 2007) . Microscopic lesions in these rainbow trout included increased erythrophagia, clusters of cellular degeneration in the hematopoietic portion of the kidney and occasionally epicarditis, endocarditis and myocarditis. These lesions are very different from the typical necrosis in liver and kidney that occur in infected Atlantic salmon, and if they were viewed in rainbow trout under field conditions, it is unlikely that ISA would have been considered as a differential diagnosis (MacWilliams et al., 2007) . Biacchesi et al. (2007) obtained 80-100% mortality of genetically susceptible juvenile rainbow trout i.p. injected with ISAV isolate Glesvaer/2/90 of European genotype. The moribund fish displayed typical pathological signs of ISA, including exophthalmos, pale gills and abdominal congestion (Biacchesi et al., 2007) . Cumulative mortalities of 95% and 20% occurred in i.p. injected Atlantic salmon and amago trout, respectively, with ISAV isolate Glesvaer/2/90 (Ito et al., 2015) . However, there was no evidence of horizontal transmission Godoy, M., Aedo, A., Kibenge, M., Groman, D., Yason, C., Grothusen, H., Lisperguer, A., Calbucura, M., Avendaño, F., Imilán, M., Jarpa, M., Kibenge, F., 2008 Chile. BMC Vet. Res. 4, 28. Figure 2B. Copyright © Panphut et al. (2011) , with permission http://www.biomedcentral.com/ download/license.pdf no changes were made. of ISAV from moribund amago trout to coreared juvenile Atlantic salmon, although ISAV was detected by RT-PCR in surviving amago trout (Ito et al., 2015) . Aamelfot et al. (2012) demonstrated that only Atlantic salmon macrophagelike cell lines (ASK-2, SHK-1 and TO) have the ISAV cellular receptor, hence, their permissiveness for ISAV replication. These authors further investigated the presence of the ISAV cellular receptor on cells from a large number of marine and freshwater fish species with a view of identifying the ISAV host range in wild fish (Aamelfot et al., 2014) . The study found that all salmonids expressed the receptor, but only some of the codlike and perchlike fish did, whereas all flat fish tested negative for the ISAV receptor (Aamelfot et al., 2014) . However, the fact that CHSE-214 cells (which are permissive to ISAV isolates of North American genotype, Kibenge et al., 2000) do not have the ISAV receptor (Aamelfot et al., 2012) suggests that ISAV may use more than one cellular receptor (ie, the natural host for ISAV may include both salmonid and nonsalmonid species). ISAV does not infect humans or other mammals since the virus is inactivated at pH values below 4.5 (and human gastric secretions are at pH 2.0) and does not replicate in vitro at temperatures of 25°C or above , and it does not appear able to replicate in human cells (Anonymous, 2000) . ISA virus is stable in both freshwater and seawater, and horizontal transmission has been demonstrated by cohabitation of healthy and ISAV-injected fish (Trojans) (Simko et al., 2000; Mikalsen et al., 2001) , with the healthy fish becoming infected by coming in contact with biological material contaminated with ISAV because of coprophagy, which in sea farms occurs due to crowding (reviewed by Cottet et al., 2011) . Transmission between aquaculture farms can be linked to fish farming practices such as movement of feed boats and well boats carrying fish, and in ballast water, sharing of personnel and equipment (nets, barges, etc.), movement of infected fish, as well as movement of virus through the water column due to proximity to fish slaughterhouse processing plants or other fish farms. Recovered farmed Atlantic salmon can become carriers; 30% of ISA outbreaks in Norway were attributed to other farms in proximity. Aldrin et al. (2011) used seaway distances between farms and genetic distances between ISAV isolates of farms in Norway with ISA between 2003 and summer 2009 to predict the rate of ISA infection of salmon farms. The fitted model predicted that the risk of infection from a neighborhood infectious farm decreased with increasing seaway distance between the two farms. Furthermore, for a given infected farm with a given ISAV genotype, the source of infection was significantly more likely to be ISAV of a small genetic distance than of moderate or large genetic distances. Nearly half of the farms with ISA in the investigated period were predicted to have been infected by an infectious farm in their neighborhood, whereas the remaining half of the infected farms had unknown sources. Sea lice (Lepeophtheirus salmonis) may serve as the mechanical vector . Migratory wild salmonids (eg, Atlantic salmon, rainbow trout, brown trout, and sea trout) may be subclinically infected and serve as carriers, particularly in virus spread over long distances (ASF, 2012) , similarly to migratory wild birds spreading avian influenza viruses. In Chile, the involvement of ISAV in a disease outbreak was officially verified in Atlantic salmon in mid-June 2007 (Godoy et al., 2008) , where all isolates that were obtained from outbreaks and had their segment 6 sequence belonging to ISAV-HPR7b, similar to isolates from Norway, but had acquired a mutation consisting of a 33 base-pair insert in their segment 5 sequence . Phylogenetic analyses of the Chilean ISAV isolates from different outbreaks suggested that the virus was introduced from Norway in 1996 , probably through fertilized salmon eggs (Vike et al., 2009; Cottet et al., 2010) . Vertical transmission of ISAV was recently reported to occur with virulent ISAV-HPRΔ (Marshall et al., 2014) , although it may be of little significance in the epidemiology of ISAV infection (Rimstad et al., 2007) . Several reports have described the main risk factors associated with fish farms getting ISA outbreaks (Jarp and Karlsen, 1997; Hammell and Dohoo, 2005; Aldrin et al., 2010; Mardones et al., 2009 Mardones et al., , 2011 . It is now well established that in Atlantic salmon, ISAV specifically targets endothelial cells lining the blood vessels in all organs (Falk and Dannevig, 1995; Koren and Nylund, 1997; Aamelfot et al., 2012) , macrophages (Moneke et al., 2003) and red blood cells (Workenhe et al., 2008; Aamelfot et al., 2012) . Thus, virus replication occurs in several organs particularly the heart, liver, head kidney and spleen, causing a generalized infection of the vascular system. Virus enters primarily via gills (Totland et al., 1996) , but it also occurs through the mouth . Atlantic salmon gill epithelial cells express 4-O-acetylated sialic acid, the preferred ISAV receptor, and the virus yield in primary gill epithelial cell cultures is comparable with that of SHK-1 cells (Weli et al., 2013) . The most extensive and prolonged virus replication is in heart tissue (Moneke et al., 2003 (Moneke et al., , 2005 . The virus is rapidly disseminated via the circulatory system (Nylund et al., 1995a; Rimstad et al., 1999) . The nonpathogenic ISAV-HPR0 replicates mainly in Atlantic salmon gills, causing only transient subclinical infection (Christiansen et al., 2011) . In contrast to the virulent ISAV-HPRΔ that targets endothelial cells, ISAV-HPR0 was shown to replicate only in the epithelial cells of Atlantic salmon gills (Aamelfot et al., 2016) . Studies on the pathogenesis of ISA showed that histopathological lesions appeared at about 12-18 days postinoculation in experimentally infected fish, coinciding with onset of mortality (Dannevig and Falk, 1994; Speilberg et al., 1995) . Moneke et al. (2005) used in situ hybridization (ISH) with a riboprobe targeting ISAV segment 7 mRNA to study the correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with ISAV. Severe histopathological lesions were observed in tissues beginning at the onset of mortality and correlated with maximum intensity and frequency of ISH signals, particularly in the liver, kidney and heart. The distribution of ISH signals indicated the presence of a viremia, as signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first to show ISH signals, and the signals lasted longest in the heart, possibly due to increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish (Moneke et al., 2005) . Kibenge et al. (2006) compared the infectivity of different ISAV isolates at a dose of 10 6 TCID 50 /0.2 mL/fish given intraperitoneally in three different farmed fishes (Atlantic salmon, coho salmon and rainbow trout) and found that the most virulent strains caused the highest mortalities in Atlantic salmon (>95% mortality) with the shortest duration (9-12 days). These highly pathogenic ISAV strains also caused mortality in rainbow trout with systemic hemorrhagic lesions and an ISH pattern with ISAV riboprobes that were consistent with those typically seen in Atlantic salmon infected with ISAV (MacWilliams et al., 2007) . Bony fish represent a transition point on the phylogenetic spectrum between species possessing only innate immunity (ie, invertebrates) and species depending heavily on adaptive immunity (ie, mammals). Studies of host response to virus infections suggest that mammals and bony fish are more or less similar in the major mechanisms of both innate and adaptive immune responses against viruses (Workenhe et al., 2010) . ISAV targets Atlantic salmon vascular endothelial cells and macrophages, is known to induce the innate type I IFN system, as well as adaptive immune response genes of Atlantic salmon (Jørgensen et al., 2008; LeBlanc et al., 2010; Lauscher et al., 2011) ; and ISAV isolates have strain-specific variations in their ability to induce immune response genes (Workenhe et al., 2009; Svingerud et al., 2013) , although the immune responses mounted do not restrict the replication of the virus (Kileng et al., 2007) . A luciferase reporter assay utilizing Atlantic salmon Mx protein and minimal IFN promoter and transient expression of ISAV proteins showed that segment 7 ORF1 and segment 8 ORF2 proteins are major-and minor-IFN system antagonizing proteins, respectively Garcia-Rosado et al., 2008) . Hetland et al. (2010) showed differences in the distribution of MHC I, MHC II and CD8 cell populations between control and ISAV-infected salmon that support the proposition that ISAV, as with influenza virus, activates CD8-positive T-cell responses. Atlantic salmon antibody response to ISAV studied by Western blotting revealed that ISAV antibodies bound exclusively to the viral nucleoprotein (Falk and Dale, 1999) and HA protein (Clouthier et al., 2002) . The discovery of antibody-mediated uptake and replication of ISAV in macrophagelike fish cell lines (Joseph et al., 2003) suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon targets endothelial cells lining blood vessels and macrophagelike cells. However, the immune adaptive response against ISAV following clinical disease appears to provide full protection against reinfection by different ISAV strains (Ritchie et al., 2009) . In contrast, it is not known if infection with ISAV-HPR0 viruses offers immunological protection against new infections with virulent ISAV-HPRΔ. The laboratory diagnostic assays for ISA include histological examination (Evensen et al., 1991; Speilberg et al., 1995; Byrne et al., 1998) and electron microscopy (Hovland et al., 1994) of fish tissues, the isolation of ISAV in SHK-1 (Dannevig et al., 1995a,b) , ASK (Rolland et al., 2005) and CHSE-214 cell lines Kibenge et al., 2000) and electron microscopic examination of a positive isolate, the use of RT-PCR Devold et al., 2000) and IFAT on positive virus isolates or on tissue samples from suspected fish (Falk and Dannevig, 1995; and ISH on tissue samples from suspected fish (Gregory, 2002; Moneke et al., 2003) . In addition, an indirect enzyme-linked immunosorbent assay (ELISA) and indirect competitive ELISA that could detect ISAV-specific antibodies in the sera of infected and vaccinated fish have been described (Kibenge et al., 2002 ). An ultrasensitive real-time nucleic acid sequence-based amplification (NASBA) method for ISAV was reported by Starkey et al. (2006) . A one-tube real-time RT-PCR for ISAV using SYBR Green chemistry was first described by Munir and Kibenge (2004) . The assay had a detection limit of 0.006 ng of ISAV RNA, could be completed in 80 min, and provided the potential to quantitate even noncytopathic virus in cell cultures. Several real-time RT-PCRs using TaqMan chemistry have been reported since then (Plarre et al., 2005; Snow et al., 2006; Workenhe et al., 2008; Christiansen et al., 2011) . Godoy et al. (2010) used the real-time RT-PCR with TaqMan chemistry to detect vRNA in formalin-fixed paraffin-embedded tissues. For the ISAV TaqMan assay, the OIE Manual of Diagnostic Tests for Aquatic Animals (OIE, 2014) recommends primer-probe sets targeting ISAV segments 7 and 8 developed by Snow et al. (2006) . Most diagnostic laboratories use the Snow et al. (2006) primer-probe set targeting segment 8 (Kibenge et al., , 2012a . Other laboratories and researchers have found the Plarre et al. (2005) primer-probe set targeting segment 7 to be more sensitive. Other researchers have published ISAV segment 8 primer-probe sets that are highly sensitive (Christiansen et al., 2011) but are not included in the OIE Manual (OIE, 2014) . Most recently, it was suggested that using ISAV segment 3 real-time RT-PCR may be a more sensitive method of virus detection and quantitation (Valenzuela-Miranda et al., 2014) . Routinely, once a clinical sample has been determined to be positive for ISAV, conventional RT-PCR with segment 6 primers ) is commonly used to obtain a PCR product that is sequenced in order to determine the HPR type and genotype of the ISAV present. Single-agent diagnostic assays such as singleplex PCR/RT-PCR that are used for pathogen surveillance/screening programs are severely limited since they are based on known pathogen nucleic acid sequence information. These assays are good in a characteristic disease outbreak or in situations suggestive of infection with one known pathogen but are not ideal in the absence of clinical signs or in situations where a particular disease is not known to occur, such as ISA in Chile prior to June 2007 (Kibenge et al., 2012a) . The methods used to control the spread of ISAV following an ISA outbreak have improved with increased knowledge about the epidemiology of ISA. For example, in 2005 the Faroe Islands dealt with ISA outbreaks by depopulating and fallowing all seawater sites and restocking under new regulatory management procedures. All countries where ISA has occurred now manage an ISA surveillance program, have restrictions on transportation and movements of farmed fish, have "all in-all out" (year-class separation or generation segregation) fish farming with fallowing periods and zone management, mandatory depopulation and disinfection of infected farms with fallowing; and they also have regulations on disinfection of wastewater from fish slaughterhouses and of intake water to hatcheries (Rimstad et al., 2007 (Rimstad et al., , 2011 Alvial et al., 2014; Aamelfot et al., 2014) . Smail et al. (2004) reported six commercially available disinfectants efficacious against ISAV for the disinfection of fish farming equipment or fish ova. The OIE, which provides international standards and guidelines for safe trade in animals and their products, has a list of notifiable aquatic animal diseases (those with a risk of spread through trade in aquatic animals and their products) that includes infection with ISAV-HPR-deleted or ISAV-HPR0 (OIE, 2015) . Because the discovery of a reportable disease is subjected to OIE trade standards, the presence of either ISA or ISAV infection often leads to expensive eradication or control efforts, and suspect populations may be quarantined until a definitive diagnosis can be made. Good management and increased biosecurity by fish farm managers in combination with early detection of infection through enhanced surveillance and use of rapid and sensitive diagnostic methods are key to preventing ISAV spread. Due to the increase in demand for international trade of live, fresh and frozen fish, areas previously unaffected by ISAV are increasingly exposed to the potential spread of the virus. The OIE currently has three levels of official confirmation of infection with ISAV (OIE, 2014): • Confirmation of ISA: The following criteria should be met: detection of ISAV in tissue preparations by IHC on fixed sections or IFAT on tissue imprints or fixed sections in addition to either (1) isolation and identification of ISAV in cell culture from at least one sample from any fish on the farm or (2) detection of ISAV by RT-PCR by the methods described in the relevant sections of the OIE Manual (OIE, 2014). The following criteria should be met: (1) isolation and identification of ISAV in cell culture from any fish sample on the farm or (2) isolation and identification of ISAV in cell culture from at least one sample from any fish on the farm with corroborating evidence of ISAV in tissue preparations using either RT-PCR or IFAT/IHC. The following criteria should be met: detection of ISAV by RT-PCR followed by independent amplification and sequencing of the HPR of segment 6 to confirm the presence of HPR0 only. Vaccination against ISAV was reviewed by Kibenge et al. (2012b) . Control of ISA by vaccination has been used in North America (since 1999 ), Faroe Islands (since 2005 , in most parts of Norway (since 2009) and in Chile (since 2010), although the currently available vaccines do not seem to offer complete protection in Atlantic salmon (Falk, 2014) . It has been shown that the level of protection with ISAV-inactivated vaccines is correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produce detectable ISAV-specific and neutralizing antibodies and have protection with a relative percent survival (RPS) of as high as 86% (Lauscher et al., 2011) . It has also been established that virus is more rapidly eliminated from vaccinated fish than from nonvaccinated fish (reviewed by Kibenge et al., 2012b) . The unprecedented 2007-11 spread of ISA in the Atlantic salmon industry in Chile created a high demand for ISA vaccines, which resulted in improved vaccine products being developed and marketed in Chile (Murias, 2012) . Up to six different vaccines, five of them being inactivated whole ISAV emulsified with adjuvant and administered by intraperitoneal injection and one oral vaccine (available for the Chilean market) are available commercially (Falk, 2014) . However, the efficacy of these vaccines in the presence of the widespread ISAV-HPR0 infections is not known. The ISAV genome is now completely sequenced, opening up the way to production of recombinant subunit and DNA vaccines against ISAV. This is bound to improve the efficacy of ISA vaccines. To date, there are two commercially available ISAV subunit vaccines (marketed by the Chilean pharmaceutical company Centrovet) based on ISAV HE protein expressed in yeast and delivered either in an oil-adjuvanted injection (monovalent or multivalent with Piscirickettsia salmonis, infectious pancreatic necrosis virus and Vibrio ordalii) or orally in feed as a bioadhesive cationic polysaccharide formulation (MicroMatrix, Harel, 2009 ) (monovalent or bivalent with P. salmonis) (Falk, 2014) . Oral vaccination using a vaccine preparation containing conserved regions of recombinant ISAV HE and F proteins expressed in Sacharomyces cerevisiae and encapsulated in MicroMatrix-induced production of IgM-specific antibodies and protected against challenge with a highvirulence Chilean ISAV isolate but was unable to induce expression of the innate antiviral Mx gene (Caruffo et al., 2016) . More innovative approaches for generation of novel and improved ISAV vaccines are continuously being undertaken. A salmonid alphavirus (SAV) replicon vaccine expressing ISAV HE was shown to offer protection against ISAV infection (Wolf et al., 2013) . Neither the ISAV F nor the ISAV M proteins were found to be protective when similarly expressed (Wolf et al., 2013) . When delivered by intramuscular injection, but not by intraperitoneal administration, the replicon vaccine provided high protection against subsequent ISAV challenge in Atlantic salmon and induced a strong innate response locally at the injection site, which may be beneficial and could warrant reduced doses and improved efficacy compared to conventional DNA vaccines (Wolf et al., 2014) . Most recently, the development of a novel reverse genetics system for ISAV (Toro-Ascuy et al., 2015) now offers a powerful tool to develop a new generation of ISAV vaccines. The use of RNA interference (RNAi), a highly conserved gene-silencing mechanism caused by dsRNA in both plants and animals (Hammond et al., 2000) , is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm (García et al., 2015) . Inactivated nonpathogenic Escherichia coli (E.coli), transformed with a plasmid that expressed a highly conserved region of the ISAV HE gene as dsRNA, which is the precursor of the RNAi mechanism, showed antiviral activity when added to infected ASK cells (García et al., 2015) . Tilapia lake virus (TiLV) is a novel orthomyxolike virus that has been shown to be the aetiological agent of a new highly lethal disease of tilapia (Bacharach et al., 2016) also referred to as "syncytial hepatitis of tilapia" (Ferguson et al., 2014) . Both wild and farmed lake tilapines (commercial hybrid tilapia Oreochromis niloticus x O. aureus, wild tilapia Sarotherodon (Tilapia) galilaeus (St. Peter's fish), T. zilli (common tilapia), O. aureus (Jordan tilapia), and Tristamella simonis intermedia) in Israel (Eyngor et al., 2014) and farmed Nile tilapia O. niloticus in Ecuador (Ferguson et al., 2014) were affected. The disease, which is characterized by waves of massive mortality in fish farms, appeared in Israel in 2009 and later in Ecuador. Interestingly, in Israel, fish morbidity and mortality are restricted to tilapia; several species reared in the same community as tilapines, including carp (Cyprinus carpio), and Gray mullet (Mugil cephalus), remained unaffected, even after long-term cohabitation (Eyngor et al., 2014) , whereas in Ecuador, only one strain of farm-bred tilapia Chitralada was affected (Ferguson et al., 2014) . Tilapia is the world's second-most-farmed fish after carp (see Chapter 1, Table 1 .2, in this book), with an estimated farmgate value of US$7.5 billion (FAO, 2014) , making TiLV a significant threat to aquaculture. TiLV was isolated using primary tilapia brain cells and E-11 cell lines (cloned subculture of striped snakehead (SSN-1) cell line), with CPE in E-11 cells consisting of syncytia formation (Eyngor et al., 2014) . The infectivity was sensitive to ether and chloroform, suggesting the TiLV is enveloped. Sensitivity of the viral genome to RNase I digestion indicated that the TiLV genome is likely ssRNA. Electron microscopic examination of thin sections of the infected E-11 cells with CPE showed enveloped icosahedral particles in the cytoplasm, measuring about 55 to 75 nm in diameter, whereas negative staining of infected cell culture supernatants purified on 25% sucrose cushions revealed virionlike structures of 75-80 nm surrounded by a thick coat (Eyngor et al., 2014) . Similarly sized viruslike particles, 60-70 nm in diameter with a 10-15-nmwide coat with surface projections, were observed ultrathin sections within the cytoplasm of hepatocytes of the infected affected fish (Ferguson et al., 2014) . Bacharach et al. (2016) used next-generation sequencing to assemble 10 contigs of TiLV genomic sequences and designed PCR primers that were used to amplify all 10 contigs from RNA extracts from infected fish and purified virus particles, followed by 5′ and 3′ rapid amplification of the cDNA end (RACE) generate the full sequence of the 10 viral genomic segments. The TiLV genome consists of 10 segments of linear, ssRNA of negative polarity, ranging in length from 465-1641 nt (see Table 19 .3) with a total molecular size of 10.323 kb (Bacharach et al., 2016) . Each segment has an open reading frame (see Table 19 -2), and the NCRs of all 10 segments have been determined; 13 nucleotides at the 5′ termini and 13 nucleotides at the 3′ termini are similar in all segments, an organization similar to other orthomyxoviruses (Bacharach et al., 2016) . The 3′ terminal 5 nucleotides and the 5′ terminal 6 nucleotides are highly conserved among the 10 genomic segments (3′-CCAAA U / A/G UU A / U C / A /U C / U C / U -------G A / G G A / U AA G / A/U AUUUGC-5′) (see Fig. 19 .7). In addition, all the 5′ ends of TiLV genomic RNA segments contain a short, uninterrupted uridine stretch (three to five bases long) (Bacharach et al., 2016) , which is the polyadenylation signal sequence in orthomyxoviruses (refer to Fig. 19 .4). Bacharach, E., Mishra, N., Briese, T., Zody, M.C., Tsofack, J.E.K., Zamostiano, R., et al., 2016 . Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. mBio 7 (2), e00431-16. Figure 1. Copyright In the disease described in Israel, affected tilapines showed pronounced ocular lesions, including opacity of the lens (cataract) advancing to ruptured lenses with uveitis or endophthalmitis, followed by buphthalmia (swelling of the eyeball) and loss of ocular functioning (phthisis bulbi), as well as skin erosions, brain hemorrhages, congestion of spleen and kidney and the presence of augmented melanomacrophage centers (Eyngor et al., 2014) . In contrast, the pathology of the disease in South America was focused in the liver and gastrointestinal tract (GIT) (Ferguson et al., 2014) rather than the central nervous system (Fig. 19.8) . The natural disease was reproduced experimentally using low-passage TiLV injected intraperitoneally FIGURE 19.8 Detection of TiLV RNA in the brain and liver of infected tilapia and infected E-11 cells by in situ hybridization and image of dead tilapia in Israel. (A and B) Brain sections of infected Nile tilapia hybridized with Affymetrix Cy3-conjugated probes (red (light gray in print versions)) of various polarities to TiLV segment 1 to detect genomic RNA (A) or mRNA (B). White arrowheads indicate hybridization signals. (C) Liver sections hybridized with Cy3-conjugated (red (dark gray in print versions)) Stellaris probes to segment 3 to detect mRNA. Nuclei are stained with DAPI (blue (light gray in print versions)). (D) Liver section stained with hematoxylin and eosin reveals multinucleated giant cells (asterisk). (E) TiLV-infected E-11 cells hybridized with Quasar 670-conjugated (red (dark gray in print versions)) Stellaris probe to segment 3 to detect TiLV mRNA. Nuclei are stained with DAPI (blue). (F) Images of confocal sections of cells in panel E were reconstituted into a three-dimensional image. (G) Dead tilapia at a fish farm in Israel. From Bacharach, E., Mishra, N., Briese, T., Zody, M.C., Tsofack, J.E.K., Zamostiano, R., et al., 2016 . Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. mBio 7 (2), e00431-16. Figure 3 . Copyright © Bacharach et al. (2016) , with permission http://creativecommons. org/licenses/by/4.0/. in tilapia; the experimental disease had a mortality of >80%, and it was 80-100% with cohabitation (Eyngor et al., 2014) . Survivors are immune to further TiLV infection (Eyngor et al., 2014; Ferguson et al., 2014) . In the Ecuador case, mortality started 4-7 days posttransfer in fingerlings approximately 3 cm long and weighing 3 g, with less than 20% surviving. Affected fish showed darkening, abdominal distension, scale protrusion and exophthalmia ( Fig. 19.9 ). Histopathology was characterized by necrosis of the GIT and a distinctive hepatocellular syncytial cell formation ( Fig. 19.10) , hence the name for the new disease, syncytial hepatitis of tilapia (Ferguson et al., 2014) . Eyngor et al. (2014) prepared a cDNA library of the TiLV genome by random priming and shotgun cloning. One of the clones was extended by 5′ and 3′ RACE reactions, resulting in 1326 bases of a putative TiLV sequence (GenBank accession no. KJ605629), which allowed the design of a PCR-based diagnostic test for monitoring TiLV infection in tilapines. Moreover, virus isolation is also possible using primary tilapia brain cells and E-11 cell line (Eyngor et al., 2014) . subsequently transferred to the grow-out ponds seemed to have better growth and feed conversion data, which could support the idea of using vaccination to prevent this disease. Control through breeding from fish that survive this viral disease also appears feasible (Ferguson et al., 2014) . 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