1 Infectious Pancreatic Necrosis Virus ARUN K. DHAR,1,2* SCOTT LAPATRA,3 ANDREW ORRY4 AND F.C. THOMas ALLNUTT1 1BrioBiotech LLC, Glenelg, Maryland, USA; 2Aquaculture Pathology Laboratory, School of Animal and Comparative Biomedical Sciences, The University of Arizona, Tucson, Arizona, USA; 3Clear Springs Foods, Buhl, Idaho, USA; 4Molsoft, San Diego, California, USA 1.1 Introduction and as a genome-linked protein, VPg, via guany- lylation of VP1 (Fig. 1.1 and Table 1.1). Infectious pancreatic necrosis virus (IPNV), the aetio- Aquabirnaviruses have broad host ranges and logical agent of infectious pancreatic necrosis (IPN), differ in their optimal replication temperatures. is a double-stranded RNA (dsRNA) virus in the fam- They consist of four serogroups A, B, C and D ily Birnaviridae (Leong et al., 2000; ICTV, 2014). (Dixon et al., 2008), but most belong to serogroup The four genera in this family include Aquabirnavirus, A, which is divided into serotypes A1–A9.The A1 Avibirnavirus, Blosnavirus and Entomobirnavirus serotype contains most of the US isolates (reference (Delmas et al., 2005), and they infect vertebrates and strain West Buxton), serotypes A2–A5 are primar- invertebrates. Aquabirnavirus infects aquatic species ily European isolates (reference strains, Ab and (fish, molluscs and crustaceans) and has three spe- Hecht) and serotypes A6–A9 include isolates from cies: IPNV, Yellowtail ascites virus and Tellina virus. Canada (reference strains C1, C2, C3 and Jasper). IPNV, which infects salmonids, is the type species. The IPNV genome consists of two dsRNAs, segments A and B (Fig. 1.1; Leong et al., 2000). Segment A 1.1.1 IPNV morphogenesis has ~ 3100 bp and contains two partially overlap- ping open reading frames (ORFs). The long ORF Two types of particles (A and B) are produced dur- ing infection. After replication, dsRNA is assembled encodes a 106 kDa polyprotein (NH2-pVP2-VP4- VP3-COOH) that is co-translationally cleaved by into the 66 nm diameter non-infectious particle A, in the VP4 (viral protein 4) protease (29 kDa) to which the capsid is composed of both mature (VP2) generate pVP2 (62 kDa; the precursor of the major and immature (pVP2) viral polypeptides. Proteolytic capsid protein VP2) and the 31 kDa VP3 (Petit processing of the remaining pVP2 into VP2 com- et al., 2000). The short ORF encodes VP5, a 17 kDa, pacts the capsid to the 60 nm diameter infectious arginine-rich, non-structural protein that is pro- particle, referred to as particle B (Villanueva et al., duced early in the replication cycle.VP5 is an anti- 2004). The VP2 protein comprises the outer capsid, apoptosis protein similar to the Bcl-2 family of while the VP3 protein forms the inner layer of the proto-oncogenes.VP5 is not required for IPNV mature virion. Additionally, VP3 remains associated replication in vivo and its absence does not alter with VP1 and VP4, as well as with the polymerase- virulence or persistence in the host (Santi et al., associated genome. 2005). Segment B has ~ 2900 bp and encodes the polypeptide VP1 (94 kDa) which is an RNA- 1.1.2 IPNV tertiary structure dependent RNA polymerase.VP1 is found both within the mature virion as a free polypeptide with Virions are non-enveloped with a T13 lattice icosa- RNA-dependent RNA polymerase-associated activity hedral morphology 60 nm in diameter, and have a *Corresponding author e-mail: [email protected] or [email protected] © P.T.K. Woo and R.C. Cipriano 2017. Fish Viruses and Bacteria: Pathobiology and Protection 1 (P.T.K. Woo and R.C. Cipriano) pVP2 RNA 1 VP2 VP4 VP3 1 VP5 467/468 773/777 1060 529/530 RNA 2 VP1 1 1045 Fig. 1.1. Genome organization of infectious pancreatic necrosis virus (IPNV). The numbers at the bottom of the segments in the diagrams indicate the amino acid number(s). pVP2 is the precursor of the IPNV viral protein VP2. RNA 1 is segment A and RNA 2 is segment B of the viral dsRNA. Table 1.1. Proteins encoded by infectious pancreatic necrosis virus (IPNV) and their functions. Protein Molecular mass Function(s) References VP1 94 kDa RNA-dependent-RNA polymerase; viral replication Calvert et al., 1991; Leong et al., 2000; Graham et al., 2011 VP2 54 kDa Major capsid protein, contains most antigenic Coulibaly et al., 2010 determinants; has a structural function VP3 31 kDa Minor capsid protein, interacts with the major capsid Leong et al., 2000; Bahar et al., protein VP2 in capsid formation, associates with the 2013 dsRNA genome, recruits polymerase into capsids, contains some antigenic epitopes VP4 29 kDa Protease involved in processing the polyprotein encoded Feldman et al., 2006; Lee et al., in segment A of IPNV dsRNA 2007 VP5 17 kDa Arginine-rich anti-apoptosis protein, similar to the Bcl-2 Magyar and Dobos, 1994; Santi family of proto-oncogenes et al., 2005 buoyant density of 1.33 g/cm3 in CsCl (Delmas was first isolated in 1960 (Wolf et al., 1960). It was et al., 2005). The viral capsid surface contains VP2 subsequently reported in Europe during the early proteins, and the three-dimensional (3D) structures 1970s and has also been reported in many other of these are known for IPNV and IBDV (Infectious countries (e.g. Japan, Korea, Taiwan, China, Thailand, bursal disease virus) (Fig. 1.2A). The IPNV VP2 Laos, New Zealand, Australia, Turkey) that are capsid is made of 260 trimeric spikes that are pro- involved either with importing salmonids or active in jected radially and carry the antigenic domains as aquaculture. IPN outbreaks are often traced to well as determinants for virulence and cellular importations and the subsequent distribution of adaptation. These are linked to VP3 in the interior infected ova/fingerlings (Munro and Midtlyng, 2011). of the virion (Fig. 1.2B). However, the spikes in IPNV are arranged differently from those in IBDV 1.3 Economic Impacts of IPN in that the amino acids controlling virulence and cell adaptation are located at the periphery in IPNV Historically, IPN is one of the top three causes of but in a central region for IBDV. The base of the spike losses in the salmonid industry. This was reflected contains an integrin-binding motif and is located in in a survey conducted by the Shetland Salmon an exposed groove, which is conserved across all Farmers Association in 2001 that showed an aver- genera of birnaviruses (Coulibaly et al., 2010). age loss of 20–30% with a cash value of 2 million pounds due to IPN (Ruane et al., 2007). From 1991 to 2002, IPN had an impact on salmon post-smolt 1.2 Geographical Distribution survival in Norwegian epizootiological studies of IPN occurs worldwide among cultured and wild sal- from 6.4 to 12.0% (Munro and Midtlyng, 2011). monid fishes. It was first detected in freshwater trout In 1998, the economic losses were estimated to during the 1940s within Canada and during the exceed €12 million (Munro and Midtlyng, 2011). 1950s within the USA (Wood et al., 1955). The virus Even today, IPN remains an important risk for 2 A.K. Dhar et al. (A) salmonid culture. For example, it was reported in 48 salmon farming facilities in Norway during 2014, although this was fewer than in previous years (Norwegian Veterinary Institute, 2015). IPN is most important in the first 6 months after sea transfer. The industry still reports significant losses due to mortalities and subsequent weakening of the surviving fish. A recent report on cumulative mor- tality in the first 6 months indicated an increase to 7.2% compared with a baseline mortality rate of 3.4%; this is more than doubling the cumulative mortality (Jensen and Kristoffersen, 2015). This same study showed that IPNV-infected cohorts challenged with other stressors showed increased levels of cumulative mortalities. For example, with pancreas disease (PD), mortality increased to 12.9%, whereas heart muscle and skeletal muscle inflammation (HSMI) increased to 16.6% when all other factors were normalized. 1.4 Diagnosis of the Infection 1.4.1 Clinical signs and viral transmission IPNV and IPNV-like birnaviruses have been isolated from salmonids as well as from non-salmonid fishes (B) (e.g. Cyprinus carpio, Perca flavescens, Abramis brama and Esox lucius), molluscs, crustaceans and pseudocoelomates (McAllister, 2007). External clini- cal signs include darkened colour, exophthalmia, abdominal distention, the presence of a mucoid pseudocast (‘faecal cast’) extruding from the vent, and haemorrhages on the body surface and at the bases of fins. Infected fish swim in a rotating manner along their longitudinal axis and death generally ensues within a few hours. Internal signs can include a pale liver and spleen, and an empty digestive tract filled with clean or milky mucus. Haemorrhages can occur in visceral organs (Munro and Midtlyng, Fig. 1.2. (A) The crystal structure of infectious 2011). IPN outbreaks characteristically consist of a pancreatic necrosis virus (IPNV) viral protein VP2 sudden increase in fry and fingerling mortalities. The showing the base domain (green), shell domain (blue) disease can also occur in post-smolts in the first and variable P domain (red). The molecular graphic was few weeks after transfer to the sea (Jensen and prepared using the free ICM-Browser software (MolSoft Kristoffersen, 2015). Stress on the host plays a key LLC, San Diego, California, downloadable at http://www. role in enhancing viral replication, mutation and molsoft.com/icm_browser.html). (B) The crystal structure even reversion to virulence (Gadan et al., 2013). The of viral protein VP1 (blue surface with residue labels) survivors of outbreaks often carry IPNV for their and the VP3 protein C-terminal (white stick and ribbon). entire lives without clinical signs.