An Overview of Betanodavirus and Perspective of Viral Nervous Necrosis (VNN) Disease in Iranian Southern Waters

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Iranian Journal of Fisheries Sciences 19(5) 2214-2233 2020 DOI: 10.22092/ijfs.2020.122274 An overview of Betanodavirus and perspective of Viral Nervous Necrosis (VNN) disease in Iranian southern waters

Ziarati M.1; Zorriehzahra M.J.2*; Kafilzadeh F.1; Kargar M.1; Ghasemi F.3

Received: January 2020 Accepted: May 2020

Abstract The Persian Gulf and its shores are important and strategic areas with a large variety of fish species. Betanodavirus infection is known to be a serious threat to susceptible fish and causing economic damages to the fisheries and fishing industry. Concerning to isolation and confirmation of VNN virus in the Mullet fish (Chelon aurata and C. saliens) in the Caspian Sea and its damages on Mullet stock, probably transmission of VNN could be hazardous in marine fish industry such as cage culture. So, the aim of this article was to characterize the distribution of the Betanodavirus in Iranian southern waters and its transmission. Finally, the issues of the transmission of Betanodavirus infection between the wild and farmed fish of different regions of the Persian Gulf is discussed. The probability of the emergence of viral epidemics and even new and virus- resistant hosts has been investigated whereby the monitoring and surveillance program for tracing the disease and the detection of the Betanodavirus presence is required before clinical signs occur in the near future. Meanwhile, screening of various species of susceptible fish and the identification of the viral carriers as a strategic approach is recommended. In fact, Eco- epidemiological studies are needed and all efforts should Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 be focused on control and prevention of probably virus contamination in the Persian Gulf and Oman Sea waters as one of the strategic points in the world.

Keywords: Betanodavirus, Persian Gulf, VNN, Iran

1-Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran 2-Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Tehran, Iran 3-Department of Animal Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran *Corresponding author's Email: [email protected] 2215 Ziarati et al., An overview of Betanodavirus and perspective of…

Introduction Sea and also, the presence of fish South of Iran has a long coastline about susceptible to Betanodavirus in the 4900 Km with the Persian Gulf and Persian Gulf, the purpose of this article Oman Sea (Harlioglu and Farhadi, was to give an overview of 2017). The Persian Gulf is the third Betanodavirus and perspective of viral largest gulf in the world and is one of nervous necrosis in Iranian Southern the most important and strategic areas waters. for its geographical and climatic characteristics (Nematzadeh, 2011). Betanodavirus Characteristics According to the latest list of Persian Betanodavirus is an icosahedral virus Gulf fish species, about 907 species are whose genome consists of two discrete belonging to 157 families are present single-stranded positive RNA (Owfi et al., 2016). The reason for this molecules (Doan et al., 2016) with no species diversity is the high salinity and poly A sequence at the end of 3’ (de la temperature (Nematzadeh, 2011), Peña et al., 2011). The RNA1 sequence which makes fishing and fisheries, as is about 3.1kb and consists of an ORF the most important and renewable coding for the RNA-dependent RNA natural resources in the region after oil polymerase (RdRp) with 110kDa, industry (Valinassab et al., 2011; which plays a vital role in the Harlioglu and Farhad, 2017). Given that replication of the virus and is also the Arabian Sea shares 87.5% of the called the Protein A. The RNA2 fish species with the Persian Gulf and is sequence (1.4kb) encodes the capsid on the other side of the Persian Gulf in protein with 36KDa (Alpha protein). In the north of the Indian Ocean and is addition, during the virus replication, a linked by the Strait of Hormuz to the subgenomic RNA is synthesized from Oman Sea and from there to the open the end region of RNA1 and is named Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 Seas (Nematzadeh, 2011; Owfi et al., RNA3 (0.4kb) (Toubanaki et al., 2015; 2016), therefore, the possibility of Valero et al., 2015; OIE, 2019). transmitting disease from one region to Betanodavirus genome organization are another, is possible. This particularly shown in Fig 1. This fragment is 370bp true for Viral Nervous Necrosis (VNN) and overlaps with the replicase ORF at especially should be considered because the nucleotide positions 2730 and 3100 it can be transferred by horizontal and (Martinez, 2015), and encodes two vertical routes by different biological unstructured proteins called B1 (111 vectors (OIE, 2019). One of the most amino acids) and B2 (75 amino acids). important emerging infectious diseases B1 boosts the antinecrotic mechanisms that affect fish throughout the world is of the host cell, while B2 is as an in fact Betanodavirus infection. Due to inhibitor protein of host iRNA, and also the spread of the virus throughout the contributes to the development of world, isolation and confirmation of mitochondrial and cellular death VNN virus in the Mullet fish (Chelon through the production of hydrogen aurata and C. saliens) in the Caspian peroxide suggesting that protein B2 is a Iranian Journal of Fisheries Sciences 19(5) 2020 2216

necrotic death factor (Doan et al., 2016; Alphanodavirus and Betanodavirus Low et al., 2017). B2 is essential for the (Mézeth et al., 2009). proliferation and infection of both

Figure 1: Betanodavirus genome organization (ViralZone:www.expasy.org/viralzone, SIB Swiss Institute of Bioinformatics)

Phylogenetic analysis of RNA2 in the Senegalese sole, gilthead sea bream, variable region T4 classifies European sea bass (Doan et al., 2016; Betanodaviruses in four different Low et al., 2017; OIE, 2019). species: SJNNV, BFNNV, RGNNV, TPNNV. In Norway, a new TNV Classification genotype was also proposed by The virus belongs to the Nodaviridae Johansen, that no isolate was obtained family, and recent studies have divided yet (Johansen et al., 2004; Korsnes, this viral family into three different 2008; Bandin and Souto, 2020). groups, including Alphanodavirus Betnaodavirus species are known to (infecting insects), Betanodavirus possess difference in growth (infecting fish), and a new third group

Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 temperatures and different hosts: called Gammanodavirus (infecting RGNNV genotype is the most abundant marine and freshwater shrimp) (Doan et and has the widest host range, and are al., 2016; NaveenKumar et al., 2017). present in the warmest water fish The viral family was named from the (optimal growth of 25-30°C) such as village of Nodamura, Japan, where the Asian sea bass, European sea bass and virus was isolated from the mosquito grouper. BFNNV is limited to cold for the first time in 1956 (Scherer and water fish species (optimal growth of Hurlbut, 1967). 15-20°C) such as Flounders, and The disease caused by Betanodavirus TPNNV infects only Tigger Puffer at an in fish has so far been named SVE (Sea average temperature of 20 °C. SJNNV bass Viral Encephalitis) (Bellance and has been detected to Japan in a limited Gallet, 1988), VNN (Viral Nervous number of warm water species (optimal Necrosis) (Yoshikoshi and Inoue, growth of 25-20°C), but over the past 1990), FVE (Fish Viral Encephalitis) decade, the virus has been found in (Comps et al., 1994) and VER (Viral Southern Europe in fish such as Encephalopathy and Retinopathy) 2217 Ziarati et al., An overview of Betanodavirus and perspective of…

(Bovo et al., 1999; Tanaka et al., 2004), and C. saliens) have continued in but now the World Organization for northern waters (Zorriehzahra et al., Animal Health (OIE) officially named 2005; Ghasemi et al., 2013; it VER on the basis of infection Zorriehzahra et al., 2013a; Nazari et al., pathology (Costa and Thompson., 2014; Ghiasi et al., 2016). 2016). The first VNN suspected case was reported in Liza klunzingeri in Persian Hosts susceptible to Betanodavirus Gulf and Oman Sea Also, some severe infection necrosis was observed in the eye and VNN has been isolated in different brain of affected Mullet and typical species (OIE, 2019). Since 1985, this vacuolation were recognized in affected virus has affected at least 120 species fish (Koohkan et al., 2014, 2015). belonging to 30 families, 11 order of marine and freshwater fish species in Types of Betanodavirus infections different geographic areas except South Acute infections: Severe clinical signs America (Curtis et al., 2001; Costa and and high mortality, Betanodavirus Thompson, 2016; Doan et al., 2016). infected cells have spread throughout Most susceptible hosts are marine the brain and retina (Martinez, 2015) fish and some species are considered without pathological changes in resistant due to lack of clinical signs. nervous tissues. However, some species such as Chronic infection: Typical histological Zebrafish and Goldfish, which were changes (Low et al., 2017) considered to be resistant to VNN from Late stage infection: Macrophage cells the beginning (Furusawa et al., 2007), are found in the brain and retina have been described as susceptible to (Martinez, 2015). VNN in recent years (Zorriehzahra et Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 al., 2013b). The highest susceptibility Geographic distribution to the disease was reported in fishes The disease has been officially reported such as groupers, flatfish, striped jack, throughout the world, with the and European sea bass. The list of exception of South America (Doan et ornamental species of freshwater al., 2016), and includes countries in susceptible to this infection is Southeast Asia (India, Indonesia, increasing (Bandin and Souto, 2020). In China, Japan, Korea, Malaysia, the future, by introducing newer aquatic Philippines, Thailand, Vietnam), species into aquaculture farming Oceania (Australia, Tahiti), the system, there is the possibility of Mediterranean Basin (France, creating new hosts (OIE, 2019). Greece,Italy, Malta, Portugal, Spain, In Iran, this disease was identified for Tunisia), the UK, Norway, the the first time in the Caspian Sea about Caribbean Islands and North America 15 years ago (Zorriehzahra et al., (USA and Canada) (OIE, 2019). 2005), and in recent years, losses from Although so far, the virus has not been this virus on the Mullet fish (C. aurata isolated from South America but it has Iranian Journal of Fisheries Sciences 19(5) 2020 2218

been detected from Amazon's healthy naturally acute infection, small amount imported fish (Martinez, 2015). of NNV was first detectable in the spinal cord at the position above swim Viral entrance portal and histological bladder in 3 out of 39 larvae examined tropism at 0-day old, then all the nervous tissues Researchers believe that the virus is a and the epithelial layer of the gill neuropathogenic agent that has a operculum, the oral cavity and the skin particular tendency towards central and of 1-day old larvae. The intensity of retinal neural tissues, and several Immunofluorescence (IFA) staining tissues have been proposed as the portal increased in nerve tissues of 2-day old of entry. One assumption is that due to larvae while it remained moderate in the proximity of the nose to the brain, the skin. By TEM observation, virions the virus first infiltrates the nasal were found either in the nerve tissues or epithelium, then reaches the brain in the hyperplastic epithelial cells of the through the olfactory nerve and skin of 1-2 day old larvae. It is ultimately reaches the retina and then suggested that the initial multiplication spinal cord (Martinez, 2015; Costa and site of NNV is in the spinal cord, Tompson, 2016). Another hypothesis is particular the area above swim bladder. that the gastrointestinal epithelium is a From this area, the virus spread primary propagation site of the virus backward to the end of the spinal cord and in fact, this region is in direct and forward to the brain, and ended in contact with water and food the retina. It is possible that the virus contaminated with the virus and from enters the host via the skin or there it easily reaches to the brain and gastrointestinal epithelium, and virus in retina through the cranial nerves the epithelium is a transient infection (Grotmol et al., 1999; Maltese and instead of productive infection. The Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 Bovo., 2007). In general, the virus is presence of NNV in epithelium could transmitted through the sensory or be a result of systemic infection or motor neuronal axon to the Central alternatively, epithelial cells of the skin Nervous System (CNS), although there may be susceptible to NNV only in the is still no evidence of the presence of very early development stage. NNV the virus in these places (Nguyen et al., exhibits neuron tropism suggesting that 1996). Although VNN has been the virus entered the spinal cord via diagnosed through its tropism to neural sensory and/or motor nerve cells linked tissues, some researchers have found to the epithelium. However, IFA evidence of its presence in other tissues positive staining was not found in the such as liver and digestive tract skin of 3-6 day old larvae during (Johansen et al., 2002; Martinez, 2015). naturally subacute infection, nor in 12- Despite the various theories of 72 hr. old larvae after NNV bath researchers, Chi (2006) raised a theory challenge. Therefore, the role of skin as based on empirical experiments and a portal of entry of SJNNV remains challenges of fish. She said that during unclear (Chi, 2006). 2219 Ziarati et al., An overview of Betanodavirus and perspective of…

Ways of transmission By the water of an infected fish or Various studies have shown that VNN infected population (Le Breton et al., can be transmitted both horizontally 1997; Hick et al., 2011). and vertically (NaveenKumar et al., Squids and subclinical infectious fish 2017). The transmission routes of that are consumed as aquatic food Betanodaviruses are shown in Fig 2 . (Gomez et al., 2010). Use of raw infectious fish for feeding Vertical transmission broodstock (OIE, 2019). It was first proposed by Breuli 1991 The use of wildlife remnants in food from Sea bass and also by Arimoto aquaculture systems (de la Peña et al., 1992 in a striped jack and in the 2011). following years also by other Fish-eating birds transmit a number of researchers. viruses from around to farmland (Kuo Typical vertical transfer methods for et al., 2012). Betanodaviruses include: Water circulating around the fish that is VNN parental transfer through infected infected with the virus (Arimoto et al., eggs 1993). It is noteworthy that, for the first time, Wild and breeding invertebrates, such the experimental method of the as snail and oysters (Gomez et al., presence of VNN in testis, the 2008; Panzarin et al., 2012). European Sea bass and Sea bream, two Artemia, rotifers and shrimp are also hosts highly susceptible to VNN and important feed for larvae in the marine sometimes asymptomatic, suggested environment and can be carriers of the that the virus could be transmitted by virus (Skliris and Richards, 1998). males too (Suebsing et al., 2012). Of Marine migratory fish transmits course, it should be noted that there is Betanodaviruses to distant geographic Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 no possibility of vertical transmission areas (de la Peña et al., 2011) of all species susceptible to VNN It has been determined that some (Martinez, 2015). larvae of infectious fish with VNN have the ability to keep infection and Horizontal transmission transmit the disease virus to the next The infected fish are an important generation trought the horizontal and reservoir of infection and viral particles vertical transmission of the (Nerland et into the water environment (Azad et al., al., 2007). 2006; Kuo et al., 2012). Iranian Journal of Fisheries Sciences 19(5) 2020 2220

Figure 2: The transmission routes of Betanodaviruses.

Viral vectors There is a lot of international trade, Water is considered the most important worms should also be considered as a abiotic vector. In the fields under risk of spreading Betanodavirus from cultivation during a clinical outbreak one region to another (OIE, 2019). from a farm section to the other part of the farm directly through water or with Clinical signs of the disease and Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 contaminated persons, boots, fishing histological lesions trips and other tools, the Betanodavirus Clinical signs of VNN in the acute and can be easily spread. In the open sea, chronic stages of the disease are due to the transmission of infection from one the proliferation of the virus and its location to another is carried out by release throughout the life cycle (Low dominant currents, farm boats and et al., 2017). Given that VNN is wildlife migration. Due to the high induced by a neuropathological agent, resistance of the virus to acidic the main clinical signs of the disease conditions and temperature of 37°C are a variety of nervous disorders such (Frerichs et al., 2000), the birds are as unusual swimming (spiral, swirling considered to be a suitable vector. On or lying down water with swollen the other hand, due to the high level of abdominal) and other neurological trade, particular attention should be symptoms such as fast and sudden paid to infected areas. The virus was swim. Specific characteristic involved identified from sand worms from the in the abnormal positioning of fish in Nereidae family near an infected farm. the water column is that the fish remain 2221 Ziarati et al., An overview of Betanodavirus and perspective of…

on the water surface. Consequently, shown in Fig 3. Other pathological non-specific signs such as loss of lesions such as swim bladder balance, loss of appetite, weight loss, inflammation, exophthalmia, skin and body color changes are observed. darkness, and anemia are also observed Probably species-specific behavioral (Adachi et al., 2008; Martineze, 2015; changes are present and different Low et al., 2017; OIE, 2019). species behave differently (Munday et al., 2002). Some clinical signs are

Figure 3: Some clinical signs of VNN in farmed Asian sea bass. A, swollen abdominal and exophthalmia; B, skin darkness.

Although the disease primarily affects cerebellum is observed (Maltese and the larval and young stages, serious Bovo, 2007). The number and size of

Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 mortality has also been reported in adult vacuoles depend on the host age and its fish (OIE, 2019). In general, younger species. Most serious lesions occur in fish are described to be more larval and younger stages and in large susceptible to the disease, as even areas of the CNS (Glazebrook et al., smaller younger fish with the same age 1990), therefore, different degrees of are more susceptible to Betanodavirus, vacuolations are seen in the brain, eye, and larger larvae are less susceptible to and spinal cord, while pyknosis and the disease as compared to their smaller karyorrhexis appear in single cells of breeds (Banerjee et al., 2014). these tissues (Munday et al., 1992). In The tissue lesions associated with the general, intracellular vacuolation in the infection of the Betanodavirus are due metencephalon and in the granular to primary neurotropism of the virus, layers of the deep retina are abundant such as the CNS and the retina. During (Galeotti et al., 1999), and pyknosis and the Betanodavirus infection, the cellular basophilia in the spinal cord vacuolation and CNS necrosis, e.g. ganglia of Oplegnathus fasciatus has telencephalon, diencephalon and been observed (Yoshikoshi and Inoune, Iranian Journal of Fisheries Sciences 19(5) 2020 2222

1990). On the other hand, inflammatory endocytosis of Betanodaviruses to host processes and the presence of cells is macropinocytosis, which uses macrophages are likely to assist in the two mechanisms: inward pit formation cellular vacuolation (Maltese and Bovo, and ruffling of the outer membrane (Liu 2007; Koohkan et al., 2014). At later et al., 2005). stages brain and eye tissues can show a lot of vacuolating and necrosis, and in The replication and release of the virus the brain of vast areas of vacuoles, they from the host showed a sponge-like appearance. Upon successful entry of the virus into According to researches, brain tissue the host cells and release of its genomic lesions vary from fish to other fish and material, the host cell biological the vacuolation is scattered in white mechanism is used and increases the matter and grey matter. Microscopic expression of genes of virus (Eckerle observations also show picnotic and and Ball, 2002; Sommerset and nuclear vacuolation of brain grey Nerland, 2004). The pathway and matter, as reported by Azad (2006) mechanism for the release of the (Azad et al., 2006; Koohkan et al., genomic material of the endosom in 2014). Betanodaviruses remain unknown (Goldfarb et al., 2006). The virus The pathogenic mechanism of +RNA genome can act as mRNA and Betanodavirus in the host body allow the expression of viral genes and The entrance to the host cell the translation of viral proteins (Bandin The necessary condition for the virus to and Souto, 2020). A non-structural enter the host cells and create a full- protein of virus called B1, coded by fledged infection is to enter into RNA3, is identified as an antinecrotic susceptible cells. There is no complete agent of cell death and increases cell Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 understanding of the biological pathway viability during the initial stages of of their binding and their cellular virus replication (Chen et al., 2009). So, receptors and the entry of the viruses to B1 protein function is very important the host, but some authors believe that during initial infection and virus binding to the acid sialic of the host cell replication (Ou et al., 2007). surface and entry through the The virus replicase, also known as endothelium occurs (Ito et al., 2008). protein A, accelerates genome synthesis Recently, there are other findings along with mitochondrial membranes suggesting the involvement of thermal and also increases the viral protein shock protein (Hsc70) in VNN binding synthesis (Su et al., 2011; Bandin and to the host cell, which serves as a Souto, 2020). In the initial stage of receptor or co-receptor to react with virus replication, protein A must be VNN capsid and plays a key role in the sufficiently synthesized to produce the early stages of infection (Chang and replication complex and RNA2 is then Chi, 2015; Costa and Tompson, 2016; used to translate capsid protein and to Low et al., 2017). The main source of regulate virion packaging (Bandin and 2223 Ziarati et al., An overview of Betanodavirus and perspective of…

Souto, 2020). The B2 is another non- Gentic lineage of the fish (Doan et al., structural protein that binds to dsRNA, 2016) which inhibits the activity of the iRNA and protecting the viral RNA from the Virus control host defense mechanism (Ou et al., After entering the viral diseases to 2007). aquatic environment, control and The release of new virions is called eradication could be difficult due to the shedding and is the last stage of a vital high stability of NNV in the aquatic virus replication cycle. Since ecosystem (Bandin and Souto, 2020). Betanodaviruses are nonenveloped Some strategies for controlling the virus viruses, the mechanism for the release include screening of embryos, water of the virus is through to be the and eggs (Kai and Chi, 2008), induction of apoptosis in the late-stage monitoring of broodstock and apply infection of the virus, which ultimately biosecurity affaires (Martinez, 2015), leads to lysis and cell death (Guo et al., washing and disinfection of larvae and 2003). fertilized eggs with ozone chlorine and use of vaccination (OIE, 2019) as well Factors influencing viral disease as applying the herbal compounds VNN infection occurs in a variety of (Saberi et al., 2017). forms, from subclinical infection to an acute infection with 100% mortality. Virus destruction Various factors can affect the pathology There are various ways to distruct the of VNN infection, which include: virus, including chemical materials Host species (formalin, binary ethylenimine, BEI, Age and size in some species of fish and β-propiolactone), physical ways (OIE, 2019) (heat and UV), and the use of certain Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 A genetic difference between viral antimicrobials and vaccines (Bandin strains and their virulence (Vendramin and Souto, 2020). The use of et al., 2013) antimicrobial peptides (AMP) has been Co-infection with other viruses as an proposed in the development of host exacerbating disease (Kokawa et al., immune response as a destructive 2008) method for infected fish (Wang et al., Environmental factors such as water 2015). Focus on advanced therapy to temperature (Iwamoto et al., 2000). stimulate the intrinsic immune system The dose of the virus (Tanaka et al., of the fish. The use of antimicrobial 1998) peptides (AMP) epinedicin-1 and Stressful factors such as population hepticidin-1 was provided for further congestion, fish nutritional survival against VNN infection characteristics and mesh size used in (Martinez, 2015). the system Several vaccines have been Inoculation route under laboratory recomended such as inactivated, conditions (Peducasse et al., 1999) Iranian Journal of Fisheries Sciences 19(5) 2020 2224

subunit, recombinant-protein, DNA and Striped Snakehead (Frerichs et al., live vaccines (Bandin and Souto, 2020). 1996) and the E-11 cell line derived Given that sometimes fish can be from SSN-1 (Iwamoto et al., 2000). contaminated with more than one genotype and there is no immune Survival outside the host response between them, multivalent Betanodaviruses are very resistant to vaccines are needed to support fish aquatic environments and can persist from different varieties (Costa and for more than 6 months in freshwater Tompson, 2016). and 6 months at 15°C (Frerichs et al., 2000), while at 25°C or higher, survival Diagnostic tests rates are dramatically negatively Virulent presence by observation of affected. Following an outbreak of tissue sections with; Transmision VNN, marine contamination with long- electron microscopy (TEM), term exposure to the virus and the Immunohistochemistry (IHC), Indirect presence of an infection source for immunofluorescence antibody (IFAT), susceptible wild species is probable. Molecular methods such as: RT-PCR, Betanodavirus easily loses its virulence RT-nPCR, RT-qPCR and Cell culture outside the water by drying and is (Moody and Crane, 2014). inactivated after a period of about 7 days at 21 °C (99%) (Maltese and In general, RT-PCR and Nested PCR Bovo, 2007). are the most accessible method for detecting a virus in clinical infectious Effective disabling methods fish but Real time PCR is also the Physical disabling by 60°C for 30 fastest, most sensitive and most specific minutes and exposed to 440 μW for ten method. Overall molecular methods minutes can be obtained while chemical Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 have advantages such as short time, inactivation can be done with sodium speed, high sensitivity and specificity, chloride (50 ppm for 10 minutes), -1 and appropriate tools for rapid detection ozone (3.0 mg Cl2L for 6/7 min) and of the virus in clinical and subclinical ethanol 60%. The virus is resistant to infectious fish. However, isolating the formalin-chloroform and ether and is virus with cell cultures following able to withstand pH conditions from 2 immunostaining and molecular to 11 for 24 hours without any loss of detection is still considered as the gold infectivity (Arimoto et al., 1996). In standard methods (OIE, 2019). A small general, common disinfectants such as number of cell lines have the ability to sodium hypochlorite, calcium isolate Betanodaviruses. Two of the hypochlorite, iodine, hydrogen most widely used cell lines for the peroxide, and benzalkonium chloride isolation of Betanodaviruses are are very useful for the inactivation the available through the European cell virus. While formalin, ether, ethanol, cultures collection of ECACC: The methanol, chloroform is not feasible to SSN-1 cell line derived from the deactivate the virus. Ozone is also used 2225 Ziarati et al., An overview of Betanodavirus and perspective of…

to prevent or reduce the infection of the mariculture in Kuwait, followed by the eggs, and on the other hand, water confirmation of the presence of the contaminated with the virus is virus in 2011, observing similar disease disinfected by exposure to UV (Frerichs in the fish in the area (Azad et al, 2005; et al., 2000; Grotmol and Totland , Azad et al., 2014). In 2014, following 2000; Costa and Tompson, 2016; OIE, an outbreak of disease in Asian Sea 2019). bass fish grown in the cage from the western coast of India, fish with clinical Discussion signs of VNN disease were tested by Due to the fact that there are numerous Nested PCR and sequencing of PCR reports of the presence of this viral reaction products, and then presence of disease in various species of fish, VNN VNN virus was confirmed (Banerjee et can be considered as a serious threat to al., 2014). the aquatic industry of the world (Zorriehzahra, 2020). VNN was reported in Iran for the first time, in the Mullet fish from the Caspian Sea (Zorriehzahra et al., 2005). Until now, the clinical signs and traces of of this disease have been observed in Iranian southern waters (Koohkan et al., 2014; 2015). On the other hand, the available evidence of contamination of fish in different parts of the world and especially countries of around of Iran like Kuwait and Bahrain, as well as Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 Figure 4: The situation of Iran and its countries such as India, can be neighboring countries in the Middle East and discussed as a ways of transmission of Persian Gulf's relationship with the Indian the virus to Iran or vice versa. In 2012, Ocean and others (https://steelguru.com).

when severe fatalities occurred in the Recent reports also indicate the Persian Gulf and the Oman Sea, presence of virus in sea bream fish from suspected samples of Liza klunzingeri the Persian Gulf waters in Bahrain. In were collected and examined fact the histopathological and mortality histopathologically. By observing the studies of VNN in different ages of the vacuolation in the brain and retina bream have identified the typical tissues, the likelihood of VNN like- vacuolation in brain and retina tissues. virus occurrence was reported This investigation was the first report of (Koohkan et al., 2014), similarly to VNN in sea bream fish in Bahrain those reported previously by Azad et al. (NaveenKumar et al., 2017). (2005) for the first time in India. In The Persian Gulf is semi-enclosed 2008, the first mortality from grouper and in the adjacent seaside basin and fish due to the VNN was reported in a north of the Indian Ocean (Owfi et al., Iranian Journal of Fisheries Sciences 19(5) 2020 2226

2016). The situation of Iran and its and Gulf. So in order to prevent the Persian Gulf's relationship with the destruction of fish stocks in the region Indian Ocean and others are shown in and before the onset of severe diseases, Fig 4. Also, the most important way is there is a pressing need for the the transmission of marine viruses screening of various species of through water and related subjects. susceptible fish and the identification of Meanwhile, vertical and horizontal the ciral carriers. Also, regarding to transmission of Betanodaviruses has develop of some marinfish industry been confirmed, therefore, in the open such as Cage Culture in the region and sea, The migration of wild fish, increase of environmental stress, some migratory birds, water flow, farm,s regional and national control and boats and the widespread trade of fish prevention approach such as could be considered as the most Survielense and Monitoring program common pathway for disease should be applied through authorized transmission (OIE, 2019). Other factors organizations strongly. that influence the composition of viral communities in marine environments Acknowledgments are the transmission of ballast water of The authors express their gratitude to ships (Kim et al., 2016), and since Iran all those involved in carrying out this is linked to neighboring countries and research, especially the professors of other open waters through the the Islamic Azad University of Jahrom waterway, it can be referred to as a and the Iranian Fisheries Science transmission way of the virus. Given Research Institute (IFSRI). that Betanodavirus has a small fragmented RNA genome and with a References possibly high mutation rate, it is Adachi, K., Ichinose, T., Watanabe, Downloaded from jifro.ir at 16:27 +0330 on Sunday September 26th 2021 possible to create a viral reassortant and K., Kitazato, K. and Kobayashi, N., infect new hosts (Costa and Tompson., 2008. Potential for the replication of 2016). On the other hand, high the Betanodavirus red spotted grouper resistance of virus in the marine nervous necrosis virus in human cell environment, as a major threat for fish lines. Archive Virology, 153(1), 15- populations (OIE, 2019). Referring to 24. the above mentioned cases and Arimoto, M., Maruyama, K. and emphasizing both the horizontal and Furusawa, I., 1993. Epizootiology of vertical transmission of Betanodavirus, Viral Nervous Necrosis (VNN) in it is possible for viral transfer in the Striped Jack. Fish Pathology, 29, 19- Iranian southern waters from the 24. Persian Gulf and the Oman Sea to the Arimoto, M., Sato, J., Maruyama, K., Indian Ocean and other open waters of Mimura, G. and Furusawa, I., 1996. the region. Since many marine species Effect of chemical and physical of the adjacent countries, such as Saudi treatments on the inactivation of Arabia, are common to the Persian 2227 Ziarati et al., An overview of Betanodavirus and perspective of…

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