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Pathognomonic of Viral Nervous Necrotic (VNN) virulence on larvae of humpback grouper (Cromileptes altivelis).


VNN attacks on aquaculture in Indonesia is still the problem especially in cultivation of marine fish (grouper). It is included one of epidemic disease almost in the world and inscribed in OIE (Office International des Epizooties) [1]. VNN spread in waters through seasonal changes of rainy to dry. VNN has attacked most of grouper cultured with mortality till 100%. Fish seeding industry also get a loss as result of VNN attack in seed stadium and even in adult because in this phase is relatively weak, resulting in an attack VNN became more acute. The symptom of VNN attack are fish whirling, happened sleeping dead or fish resides in base like death and existence of unnatural fish behavior symptom[2]. VNN causes retinopathy and encephalopathy which having wide host range. They infect grouper cells causing the decrease of the immune system in both cellular and humoral, innate and adaptive, and decline system of genes expressions of MHC II [3].

VNN is included of the family of Nodaviridae that infect fish belong to the genus Betanodavirus for which the type species is striped jack nervous necrosis virus (SJNNV) [4]. Betanodavirus is a group of viruses that do not have the envelope, small size (25-35 nm), spherical, with a genome consisting of two single-stranded positive-sense RNA molecule. The larger genomic segments (RNA1) has size of 3.1 kb and a role in encodes RNA-dependent RNA polymerase. Whereas the smaller genome segment (RNA2) has size of 1.4 kb, encodes a protein sheath in charge (protein coat) [5]. Betanodaviruses are highly resistant in the aquatic environment and able to survive for a long time in sea water at low temperatures [6].

Contamination of the aquatic environment following the appearance of an outbreak will be able to persist during long periods and represent a source of infection for wild susceptible species. In frozen fish, the virus may persist for long periods and represent a potential risk if raw fish is used for feeding [7]. Outside the aquatic environment, betanodaviruses seem to lose their cytopathogenicity very easily. In drying conditions, >99% inactivation has been observed following a 7-day period at 21[degrees]C [8].

Brain, spinal cord and retina are considered the target organs in which the virus actively replicates causing extensive tissue vacuolation. Considering the results obtained from experimental infections performed by different authors, the virus is most likely invades the host through the intestinal epithelium and peripheral nervous system, very soon reaching the central nervous tissues where it may induce the death of the host [9].

Virulence is the level of ferocity of the virus to cause disease. The level of virulence is influenced by the amount of virus, the line goes into the body of the host, the host defence mechanisms and virulence factors of the virus. Virus infection in cells and tissues always show a pathognomonic sign which is a clinical sign or lesion and characteristic of a specific disease so that it can be made a diagnosis based on simply seeing the sign or lesion[10]. One of the methods of the pathognomonic observations against the parameters of biological infected viruses are through the observation histopathology. It is one way to detect the existence of components of pathogens that are infective through observations of the micro anatomy to show an abnormal change on the level of the tissue. The purpose of this research was to analyze the tissue damage gills, liver, kidneys and intestines the larvae of Humpback grouper (Cromileptes altivelis) due to infections of the VNN (Viral Nervous Necrotic) which describe pathognomonic of VNN virulence qualitatively.

Materials And Methods

Fish Sample:

Research matter applies C.altivelis infected by VNN and control. Fish samples in stadia larvae by length 5-7 cm which infected by VNN (confirmed by PCR methods) are collected from The Brackishwater Aquaculture Development Center Situbondo (BADC/BBAP Situbondo), Indonesia.

Fish Tissue Isolation:

The eyes, brain, gills, liver, kidney and intestine of these fish infected by VNN and control were collected and fixed in 10% phosphate buffered formalin (pH 7.4) for histopathology and 2% glutaraldehyde fixation for Scanning Electron Microscope observation.


A positive sample and normal the eyes, brain, gills, liver, kidney, stomach, and intestine of these larvae C. altivelis infected by VNN were collected and fixed in 10%, phosphate buffered formalin (pH 7.4). After dehydration and clearing, samples were embedded into paraffin wax using automatic tissue processor. Paraffinated blocks were further processed in order to prepare 5 micron sections using microtome which then were stained by haematoxylin (Harris modified haematoxylin with acetic acid; Fisher Scientific, Pittsburgh, PA, USA) and eosin (10% in ethanol, Electron Microscopy Science, Pittsburgh, PA, USA) staining method and observed by light microscope.

Scanning Electron Microscope:

A positive sample and normal the eyes, brain, gills, liver, kidney, stomach, and intestine of these larvae C. altivelis infected by VNN were collected and fixed in the solution 2% glutaraldehyde fixation at a temperature of 400C. The sample is washed with phosphate buffer pH 7.4 and replaced with osmic acid fixation post solution of 1%, washed with phosphate buffer solution and dehydration. After dehidration replaced by amyl acetat abs (as a preservative until matter of time for drying), dried with a critical point in which a drying (CPD) then would on stub (holder). Coating it with a vacuum evaporator and as materials for upholstery is pure gold or carbon. Observed and captures on Scanning Electron Microscopy (SEM) by voltage of 15 kV.



Histopathological changes were observed in the eyes, brain, gill, liver, kidney and intestine (Fig 1).

No lesions were observed in any organ of the examined grouper. Brain, spinal cord and retina are considered the target organs in which the virus actively replicates causing extensive tissue vacuolation. Affected brains had pathological changes in the mesencephalon, medulla oblongata, and cerebellum. Eye lesion included vacuolation (Fig. 1A). Vacuolating retinopathy was also observed. There were large vacuoles in the granular layers of the retina, hemorrhages and necrosis in (Fig. 1B). This result show that significant histological lesions also were found in gill, hepar, kidney and intestine. Gills lesions include hypertrophy, hyperplasia, oedema, vacuolation and necrosis. The hepar exhibited generalised vacuolation, necrosis, oclussion bodies, inclussion bodies, fibriosis and cloudy swelling (Fig. 1D). There was vacuolation, hypertrophy, cloudy swelling in the kidney with generalised necrosis of haematopoietic tissue and accumulation of a hyaline substance in the lumen of kidney tubules (Fig.1E). Heart and kidney of C. altivelis are target of VNN. These organs are regulating of spreading VNN in the osmoregulation and circulation process. There were found necrosis and vacuolation in intestine (Figs. 1F).

Scanning Electron Microscope:

The observation to identify the lession on brain, eyes, gill, heart and kidney of C. altivelis using electron microscopy is shown in Figure 2.

Figure 1 show that in bud cell of retina occured virus proliferation (Fig. 2B). This lession also observed on the brain (Fig. 2E), gills (Fig.2G) and nefros of kidney (Fig.2K). There were vacuolation on eyes (Fig. 2C), hepar (Fig. 2I), and kidney (Fig. 2L). The viruses were non-enveloped, spherical to icosahedral and measured 22 to 28 nm in diameter.

The targeted attack of VNN and pathognomonic in larvae of Humpback grouper based on histophatology of tissue were summarized in Table 1. It shows that the pathogmonomic of the diseases caused by VNN was more visible in both hepar and gill tissue than other tissue.


Based on the research results, it can be known that infections cause many cell suffered damage on all the organs were observed. VNN is considered to be a serious disease of several marine fish species, characterised by significant losses associated to vacuolating lesions of the central nervous system retina, gills, liver, kidney and intestine. A typical sign of disease such as a whirling pattern of swimming was always associated with the presence of vacuoles in the central nervous system and in the retina; the lesions were correlated with the appearance of clinical signs [11]. Diseased fish of retina vacuolation could probably adapt themselves to a state of impaired vision, whereas fish with vacuolation of the entire central nervous system could not easily adapt their behaviour so as to ensure survivals [12]. On the eyes, the interaction of receptors played by laminin. Laminin is found on the surface of ephitellial and in the retina. VNN attacks can lead to exopthalmia that is the decline of the ability of the cornea. In the kidney, they will lead to the ability of the immune system declines while the brain will develop clinical symptoms such as whirling or swim play because fish balance is disturbed [13]. Characteristic lesions such as necrosis, haemorarge and vacuolation were not only observed in the central nervous system and in the retina but also in all or organ were observed. Scanning Electrone Microscope also shown that the virus was not only seen in the central nervous system and in retina, VNN at grouper are most happened at gills, kidney and liver, although at other tissue area also this virus is detected. The virus has been also detected in broodstock gonads [14-15]

Characteristic of lesions such as inclussion/occlussion bodies were observed in kidney and liver. The disease was characterized by erratic swimming behavior and hyperinflation of swim bladder; Cell infected by some virus develop inclussive/occlusion bodies within the infected cell as replication cycle progresses. There are formed as a result of an accumulation of virions or viral components, although some inclussion bodies may not contain virions.

From the observation this be explained that the occurrence of whirling, sleeping and other pathological symptoms of the dead is not only caused by an infection in the brain and retina but also in other organs. This opinion is reinforced by the discovery of Murphy et. al, (2004), that the appearance of the fish with the erratic swimming movements on fish is not only controlled by the presence of the virus in the brain but also controlled by the gills, eyes, kidneys and heart [16]. The virus can replicate and be distributed in other organs [17].

Of the results of tissue gills C. Cromileptes infected VNN showed pathological changes. Gills relate directly to the waters that many containing a virus or other material that is containing pathogens. At the time of respiration, running water causes primary lamella and secondary lamella so as to touch each other. Water that has been containing a virus directly touching lamela and went into blood capillaries, and damaged tissue through which it passes so that each the occurrence of a physical disturbance, chemically and is pathogenic originated in the neighborhood or known as injected into the body of fish will directly affecting this organs.

VNN is RNA virus having genetic matter in the form of single chain RNA (+) strand and has coat protein with molecule weigh 40-45 kD [18]. It causes the disease retinopathy and encephalopathy in grouper C. altivelis. This virus has a single strand of genetic material in the form of RNA that can directly act as a template in the synthesis of amino acids [5]. VNN does not have the envelope so that in inducing infection process of its host, VNN infect its host through an attachment immediately to the host receptor organs such as brain, heart and kidney. Subsequently, virion penetrates the cell then makes use of the metabolic machinery and pathways of the living cell to make copies of its nucleic acid and synthesisze protein subunit. There after, these basic structural components are assembled to package the new viral particles. The basic

new virions are usually from cell by lysis. The VNN immediately held an attachment to receptors which then held uncoating host the virus to insert genetic material in host cells or intracellular infection by leaving coat outside the cell [19]. The coat protein is a protein constituent VNN virion structures. Therefore, it is a crucial structure. It is not only a role in viral nucleic acid packaging VNN but also it has a primary status in the process of infection in target cell (C. altivelis). VNN coat protein have a nature to evoke cellular immune system that induce proliferation and expression of CD4, CD8, IFN-[gamma] and NFkB cell molecules in the brain, heart and kidney organs the grouper [18] [20]

One of the initial multiplication sites of the virus in larval striped jack was the spinal cord just above the swimbladder; then the virus spread backward to the end of spinal cord and forward to the brain to terminate in the retina probably via the optic nerve. Virus may gain access to the central nervous system via the peripheral nervous system, which is composed of nerves which emerge from the brain and spinal cord. Viscerosensory and visceromotor fibers connecting the sensory and effector organs of the viscera with the central nervous system may be used by the virus to reach first the brain and then the eyes, gills, liver, kidney, intestine and other organ of the fish [21].

Generally, nodaviruses fish have a strong pathogenicity to larvae and juveniles of several marine fish. maturity of the immune system could perhaps partly explain the differences in mortality values between larvae and juveniles. The lymphoid system is not fully mature at hatching; indeed, the larvae have to rely mainly on nonspecific defence mechanisms such as phagocytosis or perhaps for specific defence, on maternal immunity [22].

Besides fish stadia and the presence of virus, water is the most important abiotic vector. Betanodaviruses can be easily spread, during a clinical outbreak, by one section of the farm to another directly through the water and by contaminating personnel, nets, boots and other equipment. Adequate biosecurity measures should be established, particularly inside the hatcheries [15]. In open sea the spread of the VNN infection from one site to another is caused by tide, dominant currents, boats visiting different farms and wild migrating fish [6].


The VNN infection in grouper larvae causing damage observed in all organs with pathognomonic of diseases were visible in tissue of hepar, kidney, gill, eye, intestine and brain, the eyes and the brain that cause fish to experience whierling and irregular swimming, gills which causes sleeping dead fish, kidney which causes a decrease in immune system and intestine which causes decreased in the appetite fish.


Thanks to Ministry of Education and Culture of Republic Indonesia for supporting this research through program of Post Graduate Research Grant 2012, contract no. 0636/023-04.2.16/15/2012.


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(1) Rani Yuwanita, (2) Uun Yanuhar, (2) Hardoko

(1) Graduate Program on Fisheries and Marine Science Faculty, University of Brawijaya, Indonesia,

(2) Fisheries and Marine Science Faculty, University of Brawijaya, Indonesia

Rani Yuwanita, Uun Yanuhar, Hardoko: Pathognomonic of Viral Nervous Necrotic (VNN) Virulence on Larvae of Humpback Grouper (Cromileptes altivelis)

Corresponding Author

Rani Yuwanita, Graduate Program on Fisheries and Marine Science Faculty, University of Brawijaya, Indonesia,


Table 1. Diagnose of VNN virulence in Humpback grouper larvae

Tissue      Targetted cell   Infection   Pathognomonic     Clinical
                             of VNN                        symptoms

Eye         Bud              +           Necrosis          Whierling
            Cone             +           Vacuolation       Irregular
            Rod              +           Haemorrhage         swim

Brain       Mesencephalon    +           Vacuolation       Whierling
            ganglion         +           necrosis          Irregular
                                           Haemorrhage       swim

Gill        Melanocyte       +           Hypertrophic      Sleeping
            Limpocyte        +           Hyperplasia         dead
            Macrophage       +           Oedema
            Endothellial     +           Vacuolation
            Mucous           +           Necrosis
            Rodlet           +
            Chloride         +

Hepar       Hepathocyte      +           Necrosis

Kidney      Glomerulus       +           Necrosis          Weak
            Chromatin        +           Vacuolation
            Renal tubule     +           Cloudy swelling

Intestine   Goblet           +           Vacuolation       Decreased
            Enterocyte       +           Necrosis            appetite
            Mucous           +
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Title Annotation:Original Article
Author:Yuwanita, Rani; Yanuhar, Uun; Hardoko
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9INDO
Date:Jun 1, 2013
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