Viral Nervous Necrosis (VNN) - Betanodavirus: Molecular diagnosis (RT-PCR w/t or w/o sequencing) 

Information 28-09-15. 

Viral necrosis virus virus (VNN: Viral Nervous Necrosis) is a virus with an RNA genome belonging to the genus Betanodavirus, of the family Nodaviridae, which includes viruses that infect fishes and insects. This virus causes the disease, known as viral nerve necrosis (VNN: Viral Nervous Necrosis), also called viral encephalitis of fish or viral encephalopathy and retinopathy (VER: Viral Encephalopathy and Retinopathy), which mainly affects larvae and fingerlings of marine species, among which include grouper, cod and sea bass. However, this virus has been identified in dozens of different fish species throughout the world, both in populations of aquaculture industry species in marine farms, and in wild species. 

It is a spherical virus, with icosahedral capsid, uncoated and about 25-35 nm in diameter, and two RNA molecules ss +: RNA-1, which encodes an RNA polymerase, RNA-dependent (protein A), and RNA- 2, which encodes the capsid, in the form of two precursor proteins (α protein that will give rise to the β protein of the capsid, and the γ protein). RNA-3 is known, which is only a subgenomic transcript of RNA-1 encoding non-structural B protein. The virus is found mainly in the cytoplasm of neurons and adjacent extracellular spaces of fish affected by the disease. Cellular vacuolization and neural degeneration appear in the infected cells, observed in the brain, retina, spinal cord and nerve ganglia. The infected cells form focal aggregates in all regions of the brain, and in the nucleated layers of the retina, and their cytoplasm is replete with inclusions with a membrane containing viral particles. These lesions cause high mortality, mainly in larvae and fingerlings.

There are four main genotypes, to which a possible fifth genotype has been added:

• RGNNV (Red Spotted Grouper Nervous Necrosis virus).

• BFNNV (Barfin Flounder Nervous Necrosis virus). 

• SJNNV (Stripped jack Nervous Necrosis virus) (Caranx vinctus). 

• TPNNV (Tiger Puffer Nervous Necrosis virus). 

• TNV (Turbot Nodavirus).

This virus can have a vertical transmission and a horizontal transmission. The vertical transmission is the most important and takes place from the reproductive females infected to the larvae through the egg and the genital fluids. Horizontal transmission has been demonstrated experimentally in several ways: by putting healthy fish in contact with infected larvae; bathing fingerlings in water contaminated with tissue homogenates from fish infected with Nodavirus; adding purified viruses from diseased fish or obtained from cell cultures in SSN-1 cells. Experimental horizontal infection has been demonstrated with halibut larvae and fingerlings, but not with adults. It has been suggested that some species may be asymptomatic carriers and that larvae or fingerlings could be infected from them. Asymptomatic infection has been demonstrated seabass (Sea bass), Mozambique tilapia (Oreochronus mossambicus), or in groupers (close to marine farms).

Young fish, when infected, suffer from anorexia, change their pigmentation, and show neurological signs manifested by abnormal swimming, reaching a mortality of 100%.

These viruses, usually infect the brain of fish, eyes and spinal cord, although under stress conditions, the virus can also affect other organs such as the gonads and the digestive tract. This disease affects the nervous tissue and causes changes in infected fish that include classic signs of encephalopathy and viral retinopathy with abnormal swimming behavior (rapid and uncoordinated swimming or spiral swimming), vacuolization of the central nervous system and swollen swim bladder. Other additional signs may include lethargy, apathy and anorexia. In the larval and juvenile stages of the affected species, viral nerve necrosis (VNN) can have a 100% mortality. In adult carrier fish, the infection does not cause external signs of the disease

Currently infection by these viruses constitute one of the main problems of aquaculture, for the breeding of several species of fish, which was first described with the culture of halibut (Atlantic halibut, Hippoglossus hippoglossus), but today it affects more than 30 different marine fish species belonging to 10 families such as turbot (Scophthalmus maximus), plaice (Verasper moseri), Atlantic cod (Atlantic cod, Gadus morhua), or Pacific cod (Pacific cod, Gadus macrocephalus).

Recently, this pathology has been identified in freshwater species, such as guppies (Poecilia reticulata), and sturgeon (Acipenser gueldestaedi). 

Recommended tests for diagnosis:

Electron microscopy allows the observation of viral particles in the cytoplasm of neurons and adjacent extracellular spaces, but this is a method used in research, but it cannot be applied to the usual diagnosis.

The culture of the virus, by inoculation of tissue homogenates in a fish cell line (SSN-1) obtained from the snakehead (Snake Head, Channa striatus) could be used in laboratories specialized in fish Virology, but has the disadvantage that does not allow the replication of some strains, such as that obtained from halibut (Atlantic halibut, Hippoglossus hippoglossus).

The detection of antibodies by ELISA methods (Enzyme Immunosorbent Assay), is only of interest for infection in breeding females, but their RNA can also be detected by means of molecular diagnostic methods (RT-PCR).

Molecular diagnosis by RT-PCR (Reverse Transcriptase Polymerase Chain Reaction), is the most recommended method currently. 

Tests carried out in IVAMI:

• Molecular diagnosis (RT-PCR: Reverse Transcriptase Polymerase Chain Reaction), to detect RNA of viral necrosis virus (VNN).

Recommended sample:

• Brain tissue (at least half a brain) and one eye of the affected fish.

• The tissues of abdominal organs (ovary, bile ducts, intestine, kidneys, ...) may contain viruses, but not always (approximately 50%), which is why they are less recommended samples.

• Fish feed (in case of suspected feed infection).

Conservation and shipment of the sample:

• Refrigerated (preferred) for less than 2 days.

• Frozen: more than 2 days.

Delivery term:

• Molecular diagnosis (RT-PCR): 24 to 48 hours.

• Molecular diagnosis (RT-PCR) and sequencing: 4 to 5 days.

Cost of the test: 

• Consult: ivami@ivami.com.