FA180

Viral Nervous Necrosis (Betanodavirus) Infections in Fish1

Roy P. E. Yanong, VMD 2

Introduction The diseases caused by these viruses are commonly known as viral nervous necrosis (VNN) What are the betanodaviruses? or viral encephalopathy and retinopathy (VER). These viruses damage the central nervous system in The betanodaviruses are an important, emerging susceptible fish species and typically affect younger group of viruses known to infect over 40 marine fish stages of fish (larvae, fry, fingerlings), although species worldwide, including populations in older, market-size fish can be affected as well, with Australia, Asia, Europe, North America, Africa, and losses ranging from 15Ð100%. the South Pacific (Walker and Winton 2010). Betanodavirus infections have also been reported to Currently, there are four species of infect freshwater fish species in both natural and betanodavirus (ICTV 2009) based on genetic experimental populations. analyses: a) Striped jack nervous necrosis virus (SJNNV); b) Barfin flounder nervous necrosis virus As members of the family Nodaviridae, viruses (BFNNV); c) Tiger puffer nervous necrosis virus in the genus Betanodavirus share a number of (TPNNV); and d) Redspotted grouper nervous features that help scientists to distinguish them from necrosis virus (RGNNV). It appears that each of other viruses. These features include a small, these different species of betanodavirus may infect a non-enveloped and icosahedral-shaped capsid that number of other fish species; however, knowing surrounds genetic material made up of which strain is infecting your fish does not change single-stranded, positive-sense RNA. recommendations regarding prevention. "Non-enveloped" means that these viruses lack an outermost layer made up lipids and proteins, and the "capsid" is the protein "shell" that surrounds the genetic material of the virus. "Single-stranded, positive-sense RNA" identifies just the type of genetic material they contain.

1. This document is FA180, one of a series of the Program in Fisheries and Aquatic Sciences, School of Forest Resources and Conservation, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Original publication date December 2010. Visit the EDIS website at http://edis.ifas.ufl.edu. 2. Roy P. E. Yanong, VMD, associate professor and extension veterinarian, Tropical Aquaculture Laboratory, Ruskin, FL 33570; Program in Fisheries and Aquatic Sciences, School of Forest Resources and Conservation, UF/IFAS, Gainesville, FL 32611

The Institute of Food and Agricultural Sciences (IFAS) is an Equal Opportunity Institution authorized to provide research, educational information and other services only to individuals and institutions that function with non-discrimination with respect to race, creed, color, religion, age, disability, sex, sexual orientation, marital status, national origin, political opinions or affiliations. U.S. Department of Agriculture, Cooperative Extension Service, University of Florida, IFAS, Florida A. & M. University Cooperative Extension Program, and Boards of County Commissioners Cooperating. Millie Ferrer- Chancy, Interim Dean Viral Nervous Necrosis (Betanodavirus) Infections in Fish 2

Within what temperature range can ornamental fish the guppy (Poecilia reticulata) betanodaviruses infect fish and (Hegde et al. 2003). cause disease? In laboratory studies, medaka (Oryzias latipes) were susceptible, developing clinical signs of disease Betanodaviruses can infect tropical, sub-tropical, after betanodavirus was administered by injection or cold-temperate species. Optimal temperature (adults) and immersion (larvae) (Furasawa et al. ranges for the betanodaviruses vary somewhat 2006). Experimentally infected zebrafish (Danio depending upon the strain of the virus and the species rerio) adults and larvae also developed disease signs of fish. Optimal temperatures for SJNNV are 20Ð25¡C (68Ð77¡F); for BFNNV, 15Ð20¡C after injection with virus (Lu et al. 2008). (59Ð68¡F); and for TPNNV, 20¡C (68¡F). For What are typical external and internal RGNNV, favorable temperature ranges are signs of disease? approximately 25Ð30¡C (77Ð86¡F) (Hata et al. 2007), with growth in cell culture optimal at 25¡C Betanodaviruses more commonly cause disease (77¡F) (Ciulli et al. 2006). Upper temperature limits and mortality in larval and juvenile stages, but under for RGNNV appear to be ~32¡C (90¡F), based on the right conditions, sub-adults, market-size, and laboratory studies (Hata et al. 2007). adult fish (including broodstock) can be affected. Clinical signs reflect the fact that the nervous system Which fish species are susceptible? is targeted. In addition to mortalities of up to 100%, Betanodaviruses are primarily a concern in infected larvae and juvenile stages often show marine species and have been reported in numerous abnormal swimming behavior, including vertical species of marine food, game, and ornamental fishes. positioning and spinning; flexing of the body; and The following abridged lists are intended to show the muscle tremors. Betanodavirus causes hyperinflation diversity of susceptible species. Marine food and of the swim bladder, so diseased fish are found game fishes include red drum (Sciaenops ocellatus), primarily at the surface. Affected fish may also have cobia (Rachycentron canadum), sea bass traumatic lesions due to uncontrolled (Dicentrachus labrax), barramundi (Lates swimming/spinning. The most common clinical sign calcarifer), gilthead seabream (Sparus aurata), in adults is abnormal swimming (Bovo and Florio Pacific bluefin tuna (Thunnus orientalis), various 2008). Changes in skin pigmentation, either grouper species (Family Serranidae), and various darkening or lightening depending upon species, may flatfish species, including halibut (Hippoglossus also be seen. hippoglossus) and Japanese flounder (Paralichthys Can betanodavirus infections be oliveatus). confused with any other diseases? Marine ornamental fish species reported susceptible to betanodavirus infections include Yes, because changes in pigmentation and convict surgeonfish (Acanthurus triostegus), lined swimming behavior can be caused by other infectious surgeonfish (A. lineatus), narrowstripe cardinalfish agents (bacteria, virus, parasites) or environmental (Apogon exostigma), threespot dascyllus (Dascyllus factors including water quality (e.g., high ammonia trimaculatus), Scopas tang (Zebrasoma scopas), levels) or other toxins (e.g., pesticides). blueband goby (Valenciennea strigata), tiger puffer How do I tell if my fish has a (Takifugu rubripes), lemonpeel angelfish (Centropyge flavissimus) (Bigarre et al. 2010) and the betanodavirus infection? orbicularis batfish (Platax orbicularis) (David et al. Special diagnostic tests are required, and 2010). therefore only a fish health professional or fish Susceptible freshwater species include tilapia disease diagnostic laboratory can determine whether (Oreochromis niloticus) (Bigarre et al. 2009) and the or not your fish has a betanodavirus infection. Appropriate diagnostic tests include histopathology, Viral Nervous Necrosis (Betanodavirus) Infections in Fish 3 in which a preparation of thin, fixed-tissue sections quality parameters, nutrition, and procedural stressors are placed on a glass slide, stained, and examined is especially important. In addition, a necropsy of a under the microscope; virus isolation, which requires representative sample of fish that are showing clinical growing the virus in cell culture; testing for evidence signs of disease should be performed to rule out other of antibodies made by the fish against the virus; use contributory causes. of an electron microscope to look for viral particles themselves; and use of polymerase chain reactions Current studies suggest that betanodaviruses (PCR) or DNA probes (pieces of genetic material have the ability to infect numerous species of marine that will bind specifically to betanodaviruses) in fish and some species of freshwater fish. Spread of order to reveal evidence of viral DNA. the disease, therefore, may occur by introduction of infected fish into naïve populations. Infectivity may When a fish health professional or pathologist be increased if the immune systems of naïve fish examines tissues prepared properly for are compromised because of environmental or other histopathology, the most distinctive finding is the stressors. Older fish may also become more presence of vacuolation ("holes") (Figure 1) and susceptible to disease during periods of increased necrosis (presence of dead tissue) in the brain, spinal temperatures. cord, and retina of the eye. Inflammation may also be seen in the eye. However, in some cases Betanodaviruses may be found in a number of histopathology may not be obvious. different reservoirs. Infected fish (wild or cultured) can act as a reservoir and may be a source for net pen aquaculture species. In one study, subclinical striped jack (Pseudocaranx dentex) broodstock ("carriers") were infected with betanodavirus in multiple organs including gonads and intestines, but not within the central nervous system (Nguyen et al. 1997). This suggests that these fish, which do not show signs of the disease, may spread the virus through their gametes (eggs or sperm) or feces. Another study demonstrated evidence of betanodaviruses (RGNNV) using PCR in the tissues of several species Figure 1. Severe VNN in brain of white seabass (Atractoscion nobilis); note vacuolation ("holes" or "swiss of wild marine invertebrates, including Charybdid cheese" appearance). In normal seabass, these "holes" crab (Charybdis bimaculata), southern humpback would not be present. 500x, Nakai. shrimp (Pandalus hypsinotus), and the Mediterranean mussel (Mytilus galloprovincialis) (Gomez et al. What factors contribute to 2008). Additional research is needed to determine development and spread of whether these animals may serve as true reservoirs betanodavirus disease? and/or spread the virus to fish.

As with other viral diseases, environmental and Laboratory studies have demonstrated that procedural stressors, including high density and high betanodavirus can be spread horizontally (from temperatures, will weaken the immune systems of infected fish to uninfected, naïve fish in the same fish and make them more susceptible to infectious water body) or by exposure to contaminated water diseases including betanodavirus. Good husbandry (water that contains virus). Betanodaviruses may also may help reduce infection rates and losses. However, be transmitted through feeding of contaminated live because many different factors often lead up to a foods, including Artemia (brine shrimp), Tigriopus single disease outbreak, a complete evaluation of the japonicus (copepod), and Acetesinte medius (shrimp), facility, including review of management protocols or through feeding of raw contaminated fish. Trash (including biosecurity measures) and environmental fish species, such as Japanese jack mackerel parameters, should be carried out. A review of water (Trachurus japonicus), and mollusks, such as Viral Nervous Necrosis (Betanodavirus) Infections in Fish 4

Japanese common squid (Todarodes pacificus), fed et al. 2010; Pakingking et al. 2010). Currently, no as food were determined to be a potential source of commercial vaccines are available in the United betanodavirus (RGNNV) (Gomez et al. 2010) States.

Finally, betanodaviruses have been shown to For production facilities, washing and then transmit vertically, i.e., from broodstock to offspring disinfection of eggs after fertilization may help reduce (Breuil et al. 2002; Kai et al. 2010), potentially within infection from broodstock (if virus is on the outside or on the outside of fertilized eggs. of the egg). Use of ozone (at 1 mg O /liter for one 3 minute) for disinfection of embryos—after Does betanodavirus infection cause a fertilization and water hardening—was found to be an fatal disease? effective control method for one species of fish (striped trumpeter, Latris lineata) (Battaglene and Betanodavirus infections have been associated Morehead 2006). Laboratory tests by another with significant mortality within populations, research group with virus alone in water required sometimes as high as 100%. Larvae and juveniles are significantly less ozone (see below). typically affected, although older life stages can also succumb to infection. In addition, a number of Producers should also quarantine any new fish clinically normal fish of different species have been (and, if possible, invertebrates) in a separate building tested using molecular methods (looking for viral or area and follow appropriate biosecurity protocols RNA) and found to have evidence of betanodavirus (have dedicated equipment and use appropriate infection (i.e., are subclinical or "carriers"). disinfectants) before adding them to existing farm populations. If possible, producers should also submit Can you treat fish with betanodavirus a representative sample of fish for additional testing, infections? especially if any appear diseased or if mortalities occur. Currently there are no effective treatments for most viral infections of fish, including those caused Wholesalers and retailers should consider by betanodavirus. Depopulation followed by separating incoming fish groups by origin (e.g., disinfection is recommended. imported vs. domestic, or by country of origin) and by species, to prevent or reduce likelihood of spread, What can I do to help prevent a but additional biosecurity measures are also betanodavirus outbreak? important. Because betanodaviruses can be spread by contaminated water, use separate equipment (nets, Good husbandry and biosecurity are important, siphon hoses) for each system, disinfect systems or as is establishment of a good working relationship tanks between uses, and place footbaths and hand with a fish health professional and/or diagnostic disinfectants (alcohol spray or hand washing laboratory. If you suspect you may have fish with a stations) judiciously. Isolate sick fish if possible, and betanodavirus infection, sampling and special tests handle them separately (see the UF Extension will be necessary. Contact a fish health professional publication, "Fish Health Management for assistance. Considerations in Recirculating Aquaculture Systems," parts 1Ð3: http://edis.ifas.ufl.edu/fa099, Betanodavirus vaccines have been developed http://edis.ifas.ufl.edu/fa100, internationally and have been shown to reduce http://edis.ifas.ufl.edu/fa101). disease incidence and mortalities effectively in experimental trials. International vaccine studies have Use of a water source that may have contained shown that vaccination of larvae and older grow-out other fish or other potential sources of pathogens fish can reduce disease incidence, and that (e.g., creek, lake, or other "unprotected" water vaccination of broodstock can reduce potential source) will increase risk as well. It is safer to use transmission of virus to offspring (Lin et al. 2007; Kai water from "protected" water sources, such as deep wells, or water that has been processed with adequate Viral Nervous Necrosis (Betanodavirus) Infections in Fish 5 ultraviolet (UV) sterilization or chlorinated and Betanodaviruses can be spread from infected dechlorinated (see below). diseased fish, wild invertebrates, contaminated live or frozen foods, contaminated water, and carrier If I want to disinfect equipment after (subclinical) broodstock. Biosecurity measures for handling infected fish, what can I producers should include use of protected water use? sources, or adequate sterilization of source water; testing of potential broodstock and live or frozen Experimental studies have shown that foods; disinfection of fertilized eggs; and sampling betanodaviruses (SJNNV) can be inactivated with 50 and testing of incoming or diseased fish. Wholesalers mg/L sodium hypochlorite, calcium hypochlorite, and retailers should consider system separation of benzalkonium chloride, or iodine for 10 minutes at fish groups by origin (imported vs. domestic; 20¡C (68¡F); 60% ethanol; 50% methanol (no time importer by country; species). given); pH of 12 for 10 minutes at 20¡C (68¡F); or heat at 60¡C (140¡F) for 30 minutes. The effective Footbaths, dedicated equipment, and disinfection UV zap dose for inactivating BFNNV experimentally stations, as well as use of UV sterilization for was 1.0 x 105 µ Wsec/cm2 at a UV intensity of 440 recirculating systems will also help reduce potential µ W/cm2 in one study and 1.5Ð2.5 x 105 µ spread. Wsec/cm2 for inactivation of SJNNV in another. Ozone at 0.1 µ g/mL inactivated the virus (SJNNV) References and Suggested Reading after 2.5 minutes (Arimoto 1996; Kasai et al. 2002). Arimoto, M., J. Sato, K. Maruyama, G. Mimura, Higher doses for each of these methods may be and I. Furusawa. 1996. Effect of chemical and required depending upon organic levels, turbidity, or physical treatments on the inactivation of striped jack other differences in water quality or chemistry. nervous necrosis virus (SJNNV). Aquaculture Summary 143:15Ð22. Battaglene, S. C., and D. T. Morehead. 2006. Viral nervous necrosis (VNN), also known as Tolerance of striped trumpeter Latris lineata embryos viral encephalopathy and retinopathy (VER), is an to ozonated seawater. Aquaculture International important, emerging group of fish diseases caused by 14:421Ð429. the betanodaviruses. Numerous marine finfish and some freshwater species worldwide have been Bigarre, L., J. Cabon, M. Baud, M. Heimann, A. reported to be susceptible to the betanodaviruses, Body, F. Lieffrig, and J. Castric. 2009. Outbreak of a with mortalities as high as 100%. In many reported betanodavirus infection in tilapia, Oreochromis cases, younger life stages are affected, although older niloticus (L.), in fresh water. Journal of Fish Diseases fish are also susceptible. 32:667Ð673.

Because these viruses target the central nervous Bovo, G., and D. Florio. 2008. Chapter 4. Viral system (including the brain, spinal cord, and eye), diseases of cultured marine fish. In Fish Diseases, abnormal behavior (e.g., spinning or whirling, vol. 1, Eiras, J.C., H. Segner, T. Wahli, and B. G. abnormal posture, muscle tremors) are the most Kapoor, eds. Science Publishers, Enfield, NH. Pp. common clinical signs. Other signs include 202Ð216. darkening, traumatic lesions caused by swimming into structures, hyperinflation of the swim bladder in Breuil, G., J. F. P. Pepin, S. Boscher, and R. younger stages, and moderate to high mortalities. Thiery. 2002. Experimental vertical transmission of Since these signs can also be seen with other nodavirus from broodfish to eggs and larvae of the diseases, it is important to work closely with a fish sea bass, Dicentrarchus labrax. Journal of Fish health professional and/or fish disease diagnostic Diseases 25:697Ð702. laboratory to properly identify the cause(s) of any disease problem. Viral Nervous Necrosis (Betanodavirus) Infections in Fish 6

Ciulli, S., D. Gallardi, A. Scagliarini, M. characterization and pathogenicity studies. Archives Battilani, R. P. Hedrick, and S. Prosperi. 2006. of Virology 148:575Ð586. Temperature-dependency of betanodavirus infection in SSN-1 cell line. Diseases of Aquatic Organisms ICTV (International Committee on Taxonomy of 68(3):261Ð265. Viruses) online (Accessed November 24, 2010).

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Yanong, R. P. E. 2009. Fish health management considerations in recirculating aquaculture systems—Parts 1–3. Circulars FA-120-122. UF IFAS Cooperative Extension Service. Accessed November 14, 2010. Available on line at: http://edis.ifas.ufl.edu/fa099; http://edis.ifas.ufl.edu/fa100; http://edis.ifas.ufl.edu/fa101