Status of Emerging and Reemerging Fish Viral Infections in India

*Kollanoor Riji John, Panchavarnam Sivasankar and Mulloorpeedikayil Rosalind George*

#### **Abstract**

Aquaculture, being the fastest growing food production sector, has now become vital to the socioeconomic development of many countries. In India, aquaculture plays a significant role in food production, ensuring nutritional security, boosting agricultural exports, and generating job opportunities. The production of farmed fish has greatly expanded qualitatively and quantitatively in both freshwater and marine water regimes to fulfill the ever-growing demand. However, the occurrence of diseases is the main obstacle to sustainable aquaculture production, which has an impact on the socioeconomic status of fish farmers of the country. Viral diseases inflict irreparable damage to the aquaculture enterprise causing large-scale economic losses and ecological problems. Recently, there has been a spike in the incidence of new emerging viral diseases in diverse species of aquaculture species. Prophylactics by far being the only feasible method of viral disease control, the development of viral vaccines is highly imperative. A precise understanding of the disease pathology, etiological agent, and species susceptible to the specific diseases are highly essential in this perspective. The chapter highlights the emerging and reemerging viral diseases in the Indian aquaculture sector.

**Keywords:** aquaculture, disease emergence, virus occurrence, fish

#### **1. Introduction**

Aquaculture is one of the fastest food-producing sectors in the world that contributes significantly to the world economy. World aquaculture production has increased from 35.6 million tonnes in 2000 to 87.5 million tonnes in 2020 (**Figure 1**). At present, India is the third largest fish-producing country in the world and accounts for 7.96% of the global production. Fish production increased from 5.66 MMT in FY2000–2001 to 8.67 MMT in FY2011–2012. During FY2020–2021, the total production has been estimated at 14.73 MMT with the contribution of 11.25 MMT from inland sector and 3.48 MMT from marine sector. Indian aquaculture production during 2000–2020 ranged from about 2 million tonnes to 8.7 million tonnes in 2020 (**Figure 2**). In India, fish culture encompasses a diverse range of fishes, including Indian major carps,

#### **Figure 1.**

*World aquatic animal production increased from 35.5 million tonnes in 2000 to 87.5 million tonnes in 2020 (Source: FAO 2000–2022).*

#### **Figure 2.**

*Indian aquaculture production reached from 1943 thousand tonnes in 2000 to 8641.3 thousand tonnes in 2020 (Source: FAO 2000–2022).*

minor carps, catfishes, barbs, tilapia, climbing perch, and murrels. Additionally, due to the esthetic value and economic benefit of ornamental fish farming, it has become more and more popular throughout the world. In India, ornamental fish farming is mainly practiced in West Bengal, Tamil Nadu, Kerala, Karnataka, and states of the North East [1], and the country possesses great potential in contributing to the global ornamental sectors [2].

Intensification of aquaculture has increased productivity significantly while concurrently accompanied with several infectious diseases. Successful aquaculture production relies on various factors like stocking density, pond management, and development of the host immune system.

Health management in aquatic animals requires more attention and care for monitoring and control than terrestrial counterparts. Intensive aquaculture, in particular, has brought in more disease problems due to infectious agents, some of which are difficult to control and lead to high economic losses. By far viral diseases are found to be more difficult to control than bacterial and fungal diseases.

Until the 1980s, marine viruses were considered ecologically insignificant, because their concentrations were underestimated, but subsequent studies have confirmed that the ocean contains an abundance of organisms, including millions of virus particles per milliliter of seawater [3]. Most of the fish diseases, however, can be controlled by proper scientific management through appropriate biosecurity, nutritional adequacy, prophylactics, water and sediment quality control, adequate aeration, checking, and controlling input quality including fish seed, feed and chemicals, and constant monitoring through sampling.

New or previously unknown diseases; known diseases appearing for the first time in a new species; known diseases appearing for the first time in a new location; and known diseases with a new sign or higher virulence may be considered as emerging diseases [4]. This article provides a thorough insight into some of the important viral pathogens that are emerging and reemerging fish viruses in Indian aquaculture. Additionally, it provides current diagnostic techniques and disease control methods, as well as future directions for preventing potential diseases in wild and farmed fish.

#### **2. Common disease problems in Indian aquaculture**

Infectious diseases are the major constraints to aquaculture and the limiting factor for economic and socioeconomic development of fish farmers in India and many other countries in the world [5–7]. Some diseases have seriously affected the future development of the aquaculture sector as well as the livelihood of fish farmers. Intensification of cultural practices without the fundamental understanding of the complex balance between host, pathogen, and environment has led to many diseases that threaten present-day aquaculture [8, 9]. In India, the expansion of aquaculture into intensive and semi-intensive methods has been accompanied by an increase in production of fish and shellfish due to high stocking densities. However, stressful environmental circumstances encourage the emergence and spread of infectious diseases through variations in virulence and epidemiological factors [10]. Infectious diseases, especially viral diseases are very difficult to be controlled once established within the culture system [11] due to the peculiar environment where pathogens are constantly lurking for an opportunity when the health status of the host is compromised [12]. Many diseases in aquaculture are directly correlated with environmental deterioration including non-optimal water quality, higher microbial load, and poor nutritional status which leads to stress to the cultured animals. In addition, opportunistic pathogens present in the aquatic environment become harmful due to high stocking density [13]. Many new diseases have emerged in fish and shrimp culture as a result of expansion of the aquaculture sector, increased global movement of aquatic animals and their products, and various anthropogenic interventions in the ecosystem that lead to stress to aquatic animals.

#### **3. Emerging viral diseases of finfish**

In India, indigenous major caps (IMC) namely, catla, rohu, mrigal; exotic carps like common carp, grass carp, silver carp along with catfishes (*Clarius batrachus, Heteropneuestes fossilis, Pangassius* spp.) and freshwater prawn *Macrobrachium rosenbergii* are widely cultured. The culture of pacu, *Piaractus brachypomus*, and the exotic catfish *Pangasiandon hypophthalamus* has also grown during the past few years. Additionally, Tilapia and Pangasius offer great potential for cage culture in freshwater lakes and reservoirs [13]. Several diseases caused by viruses have been identified in fish all over the world. However, there have only been a few instances of viral diseases affecting finfish in India. Viral diseases due to Koi ranavirus (KIRV), Similar damselfish virus (SRDV), Red sea bream iridovirus (RSIV), Infectious spleen and kidney necrosis virus (ISKNV), Carp edema virus (CEV), Viral Nervous Necrosis (LCNNV-In), Tilapia Lake Virus (TiLV), and Snakehead rhabdovirus (SHRV-In) have been reported in India.

#### **3.1 Iridoviruses**

#### *3.1.1 Koi ranavirus (KIRV)*

In India, a ranavirus infecting koi (KIRV) was reported for the first time in 2015, which was isolated and characterized from moribund koi (*Cyprinus carpio*) that suffered continuous mortality exhibiting swimming abnormalities, intermittent surfacing, and skin darkening (**Figure 3**) [14]. Icosahedral virus particles of 100–120 nm were observed in the infected cell cultures, budding from the cell membrane (**Figure 4**). Sequence analysis of the major capsid protein gene showed an identity of 99.9% to that of the largemouth bass virus isolated from North America.

Iridoviruses are double-stranded DNA viruses having icosahedral capsid with a size range of about 120–200 nm and a genome size ranging from 102 to 210 kbp [15]. The family Iridoviridae is subdivided into five genera, the Iridovirus and Chloriridovirus genera that infect insects, the Lymphocystivirus and Megalocytivirus genera, which

#### **Figure 3.**

*Koi infected with koi ranavirus (KIRV) showing clinical signs such as skin darkening, loss of scales, vertical hanging, uncoordinated swimming, turning upside down, lateral rotation, intermittent surfacing, and settling at the bottom.*

*Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

#### **Figure 4.**

*Transmission electron micrographs of koi ranavirus (KIRV) in snakehead kidney (SNKD2a) cells showing icosahedral particle of 100–120 nm size.*

infect fish species and Ranavirus, which are genetically diverse and infect amphibians, fish, and reptiles [16]. Ranaviruses can cause acute, systemic disease in fish with increasing severity resulting from necrosis of kidney and spleen and hemorrhages on the skin and internal organs [16, 17]. Viruses of the genera Ranavirus are of growing concern to aquaculture owing to their ability to cause large-scale mortality in a wide variety of host species [17–20]. The common clinical signs of KIRV-infected fishes were uncoordinated swimming, rolling over, and vertical hanging before death [14].

Some iridovirus strains such as epizootic hematopoietic necrosis virus (EHNV) occur in apparently healthy fish without any clinical, indicating the carrier state. Experimental infection with EHNV also results in seroconversion in Australian frogs *Bufo marinus* without any clinical disease signs [21]. Type species FV3 of the genus Ranavirus differs from several other ranaviruses reported. Ranaviruses infect multiple cold-blooded vertebrates and have been found to undergo several host shifts suggesting the possibility of these viruses crossing the poikilothermic species barriers leading eventually to potentially devastating diseases in new hosts [22]. Molecular analysis based on the nucleotide sequences of the major capsid protein (MCP), DNA polymerase, and neurofilament triplet H1-like (NF-H1) protein gene distinguished two tropical ranavirus isolates, guppy virus 6 (GV6) and doctor fish virus (DFV) from European and Australian ranavirus isolates [23]. However, these two viruses were found to be very similar but not identical with the North American Santee-Cooper ranavirus isolated from largemouth bass [24] and KIRV [14], which had 99.21% sequence homogeneity between them for 1123 bp MCP fragment. Due to the phylogenetic variations, it is suggested that the Santee-Cooper ranavirus and related viruses such as the doctor fish virus and guppy virus may not belong to the genus [25, 26]. The presence of ranavirus was again found in carps (*Puntius sarana* and *Osteobrama belangeri*) that had extensive mortality in North East India in 2016 [27]. The sequence analysis of the 321 bp fragment has shown 98.9% homology with the major capsid protein gene of KIRV that was detected from south India [14].

#### *3.1.2 Similar damselfish virus (SRDV)*

Similar damselfish virus was isolated and characterized from marine ornamental "Similar Damselfish" (*Pomacentrus similis* Allen, 1991) in India in 2017 [28]. The virus was identified as a member of the genus *Ranavirus* of the family *Iridoviridae*. SRDV grows well in marine and freshwater fish cell lines from seabass and snakehead. It is a large and icosahedral virus of 120–130 nm having double-stranded DNA genome. Experimental infection of similar damselfish fingerlings with the SRDV showed cumulative mortalities up to 93.33%. SRDV infected fish were found to exhibit clinical signs such as skin discoloration and ulcer, lethargy, anorexia, sudden jerky movement, circling around the central axis, and settling at the bottom of the tank before mortality [28]. Phylogenetically, the virus had 99.82% identity with largemouth bass virus and 99.29% identity with KIRV across 1130 MCP fragment. Partial cross-neutralization was observed between recently isolated ranaviruses, SRDV, and KIRV against SRDV antisera indicating similarity among the immunogenic epitopes of the capsid proteins.

#### *3.1.3 Red sea bream iridovirus (RSIV)*

The red sea bream iridovirus (RSIV) is a member of the Megalocytivirus genus which causes severe mortality in farm-reared red sea bream (*Pagrus major*). RSIV infection also occurs in more than 30 other species of farmed marine fish [29, 30]. First recorded in Japan in 1990, the disease is widely distributed in several Asian countries including Taiwan, China, Hong Kong, Korea, Japan, Malaysia, Singapore, and Thailand [31]. In India, the emergence of RSIV infection was first reported in cultured Asian seabass in 2019 [32]. Affected fish were lethargic, exhibited severe anemia, petechiae of the gills, and enlargement of the spleen [33]. Histopathological changes such as increased RBC proliferation, lymphocytic infiltration, fused secondary lamellae, necrosed cellular material, and reduced secondary lamellae height was observed in RSIV-infected gill tissue. Additionally, leucocytic depopulation in the white pulp, melanomacrophage centers, increased vacuoles, and irregular intracytoplasmic viral inclusion bodies could be found in RSIV infected spleen. This virus caused 100% mortality in experimentally challenged seabass within 6 days of post-infection [32]. For RSIVD control, an effective formalinkilled vaccine was developed and is now commercially available for red sea bream (*P. major*), striped jack (*Pseudocaranx dentex*), Malabar grouper (*Epinephelus malabaricus*), orange-spotted grouper (*Epinephelus coioides*), and other fish species belonging to the genus Seriola in Japan. Complete genome analysis of the Indian strain of RSIV showed that the virus has a 111,557 bp genome and belongs to RSIV-Genotype II [34].

#### *3.1.4 Infectious spleen and kidney necrosis virus (ISKNV)*

Infectious spleen and kidney necrosis virus (ISKNV) is a type of species of the genus *Megalocytivirus* under the family *Iridoviridae* [35]. ISKNV was first detected in 1994 in the Chinese mandarin fish *Siniperca chuatsi*, that resulted in severe economic losses [36, 37]. Later, it was found spread other countries like Korea, Malaysia, Indonesia, Singapore, Australia, and Germany. The virus was reported in India in 2020 causing infection in a wide range of ornamental fish species [38]. The virus has a vast host range and can infect nearly 50 different freshwater, brackishwater, and marine species [39]. Among the popular freshwater ornamental fish species, cichlids such as the angelfish *Pterophyllum scalare*, livebearers, and some gourami species are susceptible to ISKNV [40].

#### *Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

ISKNV-infected fish showed anorexia, petechial hemorrhages, abnormal swimming, and pale coloration [37]. Gross changes included swelling of the kidneys and spleen. Histopathologically, hypertrophic cells with large basophilic cytoplasmic inclusions were found in the spleen, kidney, gill tissue, cranial connective tissue, and the endocardium [37]. Experimental infection of pearl gourami *Trichogaster leeri* and silver gourami *T. microlepis* with a tissue homogenate of pearl gourami infected by ISKNV induced 70% and 20% cumulative mortalities in the pearl and silver gourami, respectively [41]. ISKNV caused significant mortalities of ornamental fish species in India during the year 2018–2019 with various clinical signs such as erratic swimming, sluggish movement, fin rot, hemorrhage, mucus secretion, and body discoloration [32].

#### *3.1.5 Lymphocystis disease virus (LCDV)*

Lymphocystis virus disease affects a large number of freshwater, brackish water, and marine fish species across the world. LCD is characterized by epithelial benign tumors resulting from hypertrophied fibroblast connective cells of the body and fins. Presence of epidermal papilloma like benign tumor used to appear on farm-reared adult grass carp, *Ctenopharyngodon idella* in the northern part of India during the winter months (November–January) of 2017–2019. Investigation was the first report of the occurrence of lymphocystis disease in grass carp in India [42]. The viral agent was identified as LCDV LRI-18 following molecular and histological identification techniques. Phylogenetic examination of the partial nucleotide sequence of LCDV DNA polymerase gene revealed close relatedness of LCDV LRI-18 (GenBank No. MK347473) and the LCDV strain from Israel, sharing 99.0% and 96.5% homology among the respective nucleotide and amino acid sequences.

Single or multiple intracytoplasmic inclusion bodies have been noticed on lymphocystis and erythrocyte cells of grass carp infected with LCDV LRI-18. The virions were icosahedral in shape with an electron-dense core and had a size of 280 nm diameter [42]. Histopathologically, LCDV-infected grass carp had extensive hypertrophy and lamellar fusion, hemorrhage in the eye, liver necrosis, myocardial inflammation, fused intestinal villi, and glomerular degeneration. Experimental dip infection in 0.45 m filtered LCDV crude suspension did not reproduce the disease in grass carp fingerlings., and also no death was noted.

#### **3.2 Carp edema virus (CEV)**

Carp edema virus disease (CEVD) is an emerging disease of concern to koi enthusiasts and carp aquaculture around the world. Carp edema virus is a large, doublestranded DNA virus belonging to the poxvirus family of viruses (*Poxviridae*). Carp edema virus disease/koi sleepy disease differs widely from another similar disease referred to as "carp pox," which is caused by a herpesvirus (Cyprinid herpesvirus 1) that is responsible for wart-like growths on the skin in common carp varieties [43]. The CEV was first detected in Japanese koi in the 1970s and derived its name from causing edematous skin lesions in the affected fish [44]. The infection of CEV has been reported in three continents, Asia, North America, and Europe, particularly from Germany, India, China, Korea, and Iraq from common carp and koi carp [45]. The infection with CEV was reported in India for the first time in koi that was showing clinical signs similar to sleepy disease [45]. Of late, large-scale mortality caused by CEV in koi carps (*C. carpio koi*) has been found in the ornamental fish farm of Odisha, India [46].

Common carp (*C. carpio*), especially koi, can contract the carp edema virus, which can lead to disease and high mortality rates. The disease was formerly known as "viral edema of carp" because sick fish may have erosive or hemorrhagic skin lesions along with swelling (edema) of the underlying tissues (**Figure 5**) [47]. The infected fish could also exhibit the clinical signs of ulcers on body, massive necrosis of gills, and sleeping at the bottom of tanks before death (**Figure 6**) [45]. In the early stages of the disease, the gill epithelial cells at the tips of the gill filament proliferate, resulting in a thickening or "clubbing" appearance (**Figure 7**) [48]. In CEV-infected fish, the proliferation may extend to the base of the gill filament and impair gill function. Common carp and koi (*C. carpio*) are the only known susceptible species [49].

The disease is also known as "koi sleepy sickness" (KSD) due to the strange behavior of affected fish, which includes being unresponsive and lethargic and frequently

#### **Figure 5.**

*Koi infected with carp edema virus having dropsy showing internal hemorrhage and serosanguinous fluid in the peritoneal cavity.*

#### **Figure 6.**

*CEV infected the clinical signs of ulcers on body, massive necrosis of gills, and sleeping at the bottom of tanks before death.*

*Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

#### **Figure 7.**

*CEV-infected koi gill epithelial cells proliferated at the tips of the gill filament, resulting in lamellar fusion and thickening or "clubbing" appearance.*

lying motionless at the tank floor for extended periods of time if undisturbed. Once the "sleepy" carp is disturbed, they swim for a little while and soon become passive and settle on the tank's bottom [48]. The severity of the disease is greatest in juveniles, which may hang just under the surface of the water before succumbing, while adult fish may lie motionless on the bottom of the pond/tank [49].

#### **3.3 Viral nervous necrosis (VNN)**

Fish nodaviruses, members of the genus *Betanodavirus* under the family *Nodaviridae*, are the causative agents of a highly destructive disease in approximately 150 species of marine finfish species worldwide [50]. Following the first outbreak of the viral nervous necrosis disease at early larval stage in seabass hatcheries in Martinique, the French Mediterranean [51] and Queensland [52], the causative agent was first identified as a member of the family *Nodaviridae* by molecular analysis of the purified virus from infected hatchery-reared larvae of striped jack *P. dentex* [53]. Viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER), is a disease caused by nodaviruses, which are icosahedral in shape having a size range of 25–34 nm (**Figure 8**). The virus has a single-stranded bipartite positive sense RNA genome. Although adults can be affected, hatchery-reared larvae and juveniles are primarily affected by VNN outbreaks, which can cause high mortalities of up to 100% [54]. The virus was first isolated in cell culture from seabass fry using striped snakehead cell line [55]. There are four genotypes recognized under the genus including red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and striped jack nervous necrosis virus (SJNNV) based on the comparative sequence analyses of the coat protein genes [56].

The VNN has been reported in many countries including Southeast Asia (India, Indonesia, China, Japan, Korea, Malaysia, Philippines, Thailand, and Vietnam), Oceania (Australia, Tahiti), the Mediterranean Basin (France, Greece, Italy, Malta, Portugal, Spain, and Tunisia), the UK, Norway, the Caribbean Islands, and North America (USA and Canada) [50, 57]. In India, betanodavirus infection has been observed in both cultured and wild population of brackishwater/marine

#### **Figure 8.**

*Transmission electron micrographs of Lates calcarifer nervous necrosis nodavirus in SSN1 cell line.*

fish species (**Figure 9**) such as *Lates calcarifer, Rachycentron canadum, Trachinotus blochii, Mugil cephalus, Liza parsia, Chanos chanos, Epinephelus tauvina, Sardinella longiceps, Amblygaster clupeoides, Thrissocles dussumieri, Leiognathus splendens, Upeneus sulphureus,* and *Mystus gulio* (reviewed by Jithendran et al. [58]. In addition, the infection has also been recorded among aquarium fishes including *Carassius auratus* (Gold fish), *Epalzeorhynchos frenatum* (Rainbow shark), *Danio rerio* (Zebra fish) and *Amphiprion sebae* (Clown fish) [58]. *Betanodavirus* genotypes show different optimal growth temperatures, 15–20°C for BFNNV, 20°C for TPNNV, 20–25°C for SJNNV, and 25–30°C for RGNNV [59]. The temperature sensitivity of betanodaviruses seems to be regulated by the region encoding the amino acid residues 1–445 of RNA1 [59].

**Figure 9.** *Viral nervous necrosis (NNV)-infected juvenile seabass.*

#### *Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

Clinical signs due to VNN infection depend on the fish species, biological stage, phase of the disease, and temperature. However, common signs are abnormal swimming behavior (spiral swimming, whirling, horizontal looping, or darting) and loss of appetite among affected fish [60, 61]. Other signs include swim bladder hyperinflation and coloration abnormalities (pale or dark). Histopathologically, the fish show extensive necrosis of the central nervous system (CNS), with extensive vacuolation and neural degeneration of the brain as well as vacuolation of the retina [60–62]. The main clinical signs of NNV infection in freshwater fish include anorexia, descaling, and settling the bottom with dropsy [58]. An experimental infection performed using guppy (*Poecilia reticulata*) showed clear clinical signs associated with significant mortality since 15 dpi [63]. The cumulative mortalities reached up to 100% at 30 dpi in the study. A pathogenicity study in seabass fingerlings using betanodavirus revealed nervous necrosis in retinal cells following a 21-day challenge trial (**Figure 10**) [64]. Both horizontal and vertical transmission has been demonstrated in several fish species [50].

#### **3.4 Tilapia lake virus (TiLV)**

Tilapia Lake Virus (TiLV) disease is an emerging and transboundary disease of tilapia, causing mortality up to 90% globally in farmed tilapia over the last 4–5 years [65, 66]. TiLV is an enveloped, negative-sense, single-stranded RNA virus (-ssRNA) with a 10,323 kb genome and a size range of 55–100 nm diameter [65]. It was first identified as an orthomyxo-like virus and the only member of the genus *Tilapinevirus* in the family *Amnoonviridae* [65, 67, 68]. Until 2009, there were no reports on viral diseases in Tilapia. However, large-scale mortalities were seen in both wild and farmed hybrid tilapia (*Oreochromis niloticus* × *O. aureus*) in Israel during the summer of 2009, the etiological agent of which was identified as Tilapia Lake Virus (TiLV) in 2013 [65]. Further, outbreaks of TiLV have been reported from other countries

#### **Figure 10.**

*H&E stained section of the retina of experimentally infected seabass juveniles showing extensive vacuolation of the cells in outer plexifrom and inner nuclear layer. Photo courtesy: Lekshmi Haridas.*

namely, Ecuador, Colombia, Egypt, Thailand, Chinese Taipei, Malaysia, Bangladesh, Uganda, Tanzania, Peru, Mexico, Philippines, Indonesia, and USA. In India, TiLV was first reported following the outbreaks of a fatal disease in farmed tilapia in two states, West Bengal and Kerala [69].

There are several clinical signs associated with TiLV; however, corneal opacity may be one of the overt clinical signs of TiLV infection [65]. The infected fishes could exhibit other signs like anorexia, poor body condition, severe anemia, bilateral exophthalmia, skin abrasion and congestion, scale protrusion, and abdominal swelling (**Figure 11**) [70–72]. In addition, clinical signs like pale coloration of the body, gathering at the bottom, sluggish movement, abnormal swimming, and avoidance of schooling before death have also been observed during the outbreak [73]. The brain and liver are the most targeted organs for TiLV infection [66]. Histopathological changes in the liver are characterized by hepatic syncytia and thus the name "syncytial hepatitis" (**Figure 12**). Pathologies in the brain included blood vessel congestion and infiltration of lymphocytes, which have been associated with the clinical sign of irregular swimming [74]. Recently, co-infection of bacteria, *Lactococcus garvieae* with TiLV and associated mass mortality in Nile tilapia was reported in India [75].

#### **3.5 Snakehead rhabdovirus**

Rhabdoviruses and aquabirnaviruses are among the most potentially harmful viral pathogens of fishes. The family *Rhabdoviridae* comprises bullet-shaped enveloped viruses that are classified into 18 genera and 134 species (**Figure 13**) [76]. Rhabdoviruses have an enveloped virion with the nucleocapsid (30–70 nm in diameter) containing a single molecule of linear, negative-sense ssRNA genomes of 10–16 kb size, which encodes five virion structural proteins: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the polymerase (L) [77]. Rhabdoviruses infecting fish are included in the genera *Novirhabdovirus,* 

#### **Figure 12.**

*Histopathological section of TiLV-infected liver stained in hematoxylin and eosin showing putative intracytoplasmic inclusion body (arrowhead) with giant cell having multiple nuclei (arrow). Photo courtesy: Lekshmi Haridas.*

#### **Figure 13.**

*Transmission electron micrographs of snakehead rhabdovirus particles (SHRV-In) in the virus-infected SSN1 cells revealing bullet-shaped virus particles (bar 200 nm).*

*Sprivivirus, Perhabdovirus,* and *Vesciculovirus.* Rhabdovirus has been reported to cause acute disease in Rio Grande Perch (cichlid) and North American (Mexican) cichlid (*Cichlasoma cyanoguttatum*) [78]*.* These viruses are responsible for infections in a wide range of freshwater and marine fishes (*Anguilliformes, Clupeiformes, Cypriniformes, Gadiformes, Perciformes, Pleuronectiformes*, and *Salmoniformes*) [79].

Infections with three rhabdoviruses such as infectious hematopoietic necrosis (IHN), viral hemorrhagic septicemia (VHS), and spring viraemia of carp (SVC) are listed by OIE as notifiable diseases [31]. Fish rhabdoviruses generally show tissue

**Figure 14.** *Snakehead fish with surface ulcerations and hemorrhagic areas infected with snakehead rhabdovirus.*

tropism to kidney, spleen, and brain while liver, heart, and gill are also found to be the multiplication sites albeit with low titers [80]. Rhabdoviruses were isolated from infected snakeheads (*Ophicephalus striatus*) from various Southeast Asian countries such as Thailand, Myanmar (Burma), and Philippines in 1980s and also from other epizootic ulcerative syndrome (EUS)-infected fishes [81–83]. Striped snakehead skin ulcerative disease reported in striped snakehead in Burma and Thailand caused large, deep ulcerations of the skin on the head and body of fish [82, 84]. Infected fish show signs of lethargy leading to mortality within 1 week [79]. Recently, isolation of a snakehead fish vesiculovirus (SHVV) was reported from hybrid catfish in China [85]. First report of emergence of a rhabdovirus (SHRV In) in India was from an infected snakehead that were undergoing mortality in the extensive type aquaculture tanks in the rural areas of South India that had behavioral abnormalities and surface ulcerations (**Figure 14**) [86]. Experimental infection with SHRV-In exhibited clinical signs of listlessness, uncoordinated spiral swimming, anorexia, lethargy, loss of scales, discoloration, small pale white patches on the body, hemorrhagic areas, and surface ulcers in juvenile snakeheads. Internally, fish showed hemorrhages and ascitic fluid [86].

#### **4. Control measures**

For successful aquaculture production, health management in the nursery, and grow-out culture is essential because the quality and quantity of fish produced depend on biosecurity and health management measures adopted by the aquaculturist. In general, fish are stocked at high densities in nurseries and grow-out farms and therefore, improper management can lead to weak fingerlings with low survival. Prevention is always considered as the first step in controlling infectious diseases in aqua farms. General biosecurity measures like sanitizing hands before handling fish, having foot dips with disinfectants at all entry points in the farm, disinfecting the

source water are more important to prevent the frequent occurrence and spread of disease. Development of suitable preventive and control measures, specifically vaccines, immunostimulants, herbal extract, and probiotics are also of high significance, for the fish farmers to protect their crop against pathogens (**Tables 1**–**3**).


#### **Table 1.**

*Commercially available vaccines for viral diseases reported in India.*


#### **Table 2.**

*Experimental immunostimulants studied for fish viruses.*


#### **Table 3.**

*Probiotic bacteria used against viruses of fish.*

#### **5. Recent significant advances in research on the management of viral diseases**

While there are successful vaccines available for many existing viral diseases, for emerging viral infections, development of vaccines is only in the preliminary stages [87, 88]. Since there is no commercial vaccine or effective antiviral treatment against SGIV infection currently, a high-throughput in vitro cell viability-based screening assay has been developed to find antiviral compounds against SGIV using the luminescent-based CellTiter-Glo reagent in cultured grouper spleen cells by quantificational measurement of the cytopathic effects induced by SGIV infection [89]. Aqueous preparations of the medicinal plant, *Viola philippica* has been found to have excellent inhibitory effects against Grouper iridovirus GIV during the viral infection stage of binding and replication in host cells [90]. Against SVCV, a total of 35 arctigenin derivatives have been synthesized and tested for their antiviral efficacy in EPC cells. Out of 35 derivatives

#### *Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

screened, 32 were found to have a potential anti-SVCV effect at relatively high concentrations [91]. Additionally, a new coumarin derivative called 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) and imidazole coumarin derivative, 7-(4-benzimidazolebutoxy)-coumarin (BBC) have been synthesized and evaluated for the antiviral activity against spring viraemia of carp virus (SVCV), which showed that D5 and BBC had a strong antiviral activity SVCV expression in the host cells and in zebrafish [92–94]. Arctigenin (ARG) has also been found to have the highest inhibition on SVCV replication [95]. A novel coumarin derivative (C3007) could have significant potential for use as a therapeutic agent in aquatic systems and may also be appropriate for use in pond aquaculture environments to prevent viral transmission [96].

The arctigenin-imidazole hybrid derivative-15 with an eight-carbon atom linker length greatly suppressed apoptosis and the cellular morphological damage brought on by infectious hematopoietic necrosis virus (IHNV) in addition to decrease replication [97]. A new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole) octyloxy) arctigenin (EOA), significantly decreased cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum correct as cyprini (EPC) cells. In addition, it significantly inhibited apoptosis induced by IHNV in EPC cells [98].

Since broad-spectrum water-immersion antiviral treatments are highly desirable, light-activated antivirals that target the viral membrane (envelope) of viruses have been developed to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells [99]. An extract from *Ecklonia cava* has also been demonstrated for its ability to suppress VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Additionally, oral administration of the *E. cava* extract to the olive flounder increased the antiviral immune response and the efficacy of protection against VHSV, leading to the development of an antiviral status in the olive flounder [100]. Alpinone has been found to have in vitro antiviral activity against the infectious salmon anemia virus [101].

Drugs such as ammonium chloride and chlorpromazine hydrochloride drugs could be used for controlling nodavirus infection in aquaculture [102]. One of the best tools to prevent virus spread is the development of suitable vaccines. Binary ethylenimine (BEI) inactivated vaccine against the nervous necrosis virus has been generated and conferred partial protection to *Senegalese sole* when administered by intra-peritoneal injection, although they induced a different immune response [103]. An epitope-based vaccine (EBV) has also been developed using a computation approach for the first time and tested against seven banded grouper nervous necrosis virus [104].

Common disinfectants such as iodine, sodium hypochlorite, hydrogen peroxide, and formalin can be effectively used to reduce viral loads [105]. Therefore, the proper use of such disinfectants may be encouraged and put into practice in order to reduce the development of TiLV in aquaculture farms and related facilities. β-propiolactone inactivation of viral particles exhibited higher protection efficacy against virus challenge than formaldehyde [106]. When combined with the adjuvant Montanide IMS 1312 VG and booster immunizations, the β-propiolactone-inactivated vaccine provides a high level of protection from TiLV challenge in tilapia.

#### **6. Conclusion**

In India, aquaculture plays a key role in increasing production of highly nutritious and cheap protein to the masses while at the same time generating more employment opportunities. Emerging viral diseases of various categories are, however, causing great concern to the sector threatening the sustainability of aquaculture operations. A definite understanding of the etiological agents is therefore of paramount importance in generating interventions in controlling the diseases. Adequate diagnostic protocols and ability to detect different strains arising out of mutations resulting in emergent infections are also highly indispensable. Many of the viral disease agents detected in India showed variations from their global counter parts. It would be therefore essential to develop proper surveillance methods and systematic approach of documentation coupled with fast responsiveness to a reported infection to keep the emerging viral diseases under control. This would help in containing the infection and probably eliminating the incriminating agent if proper biosecurity principles are applied. Adequate care also needs to be given to the seed production of the candidate species by screening the brood stock so that the possible vertical transmission can be prevented. As the therapeutic measures are having little success with viral infections in fish, prophylactic measures, including vaccines, immunostimulants, herbal extracts and probiotics to protect the crop against viral diseases are to be extensively explored.

#### **7. Future perspectives**

Realizing the trend of increasing viral infections in fish, it is necessary to build adequate preventive measures to contain the spread of the disease. The requirements in this aspect include:


*Status of Emerging and Reemerging Fish Viral Infections in India DOI: http://dx.doi.org/10.5772/intechopen.109012*

#### **Author details**

Kollanoor Riji John1 \*, Panchavarnam Sivasankar2 and Mulloorpeedikayil Rosalind George1

1 Kerala University of Fisheries and Ocean Studies, Kochi, India

2 Fisheries College and Research Institute, Thoothukudi, Tamil Nadu, India

\*Address all correspondence to: rijijohn@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## *Edited by Shailendra K. Saxena*

This book offers a comprehensive overview of recent trends in viral outbreaks, covering general concepts of immunobiology of infections, viral outbreaks, pathology, virology, viral genomics, viromes, and antiviral pharmacology. It also discusses current clinical recommendations for managing various viral diseases, highlighting ongoing issues, recent advances, and future directions in diagnostic approaches and therapeutic strategies. The book focuses on various aspects and properties of viral outbreaks, whose in-depth understanding is crucial to protect humanity from further losses of resources and economies due to viral diseases. It provides an overview of recent developments in the detection, monitoring, treatment, and control of several viral diseases that have caused recent epidemics or pandemics.

Published in London, UK © 2023 IntechOpen © Gilnature / iStock

Viral Outbreaks - Global Impact and Newer Horizons

Viral Outbreaks

Global Impact and Newer Horizons

*Edited by Shailendra K. Saxena*