Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

Abstract

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.

Betanodavirus of Marine and Freshwater Fish: Distribution, Genomic Organization, Diagnosis and Control Measures

“…Immunohistochemistry study reveals the location of immunopositive cells and shows that the virus favours neural cells which are in the early stages of proliferation. This indicates that the virus enters the CNS along nerves and blood vessels during the viraemic stage of the disease [64,71]. The virus also can be detected by immunofluoresence microscopy by using monoclonal antibody [101].…”

“…Betanodavirus is a distinct group from the insect nodaviruses (Alphanodaviruses) [90] in the 7th report of the International Committee on Taxonomy of Viruses [8,86]. The recognized members are: barfin flounder nervous necrosis virus (BFNNV), red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV) and tiger puffer nervous necrosis virus (TPNNV) and RGNNV is most widely distributed [64,90,91,107].…”

“…Diseased fish show various clinical symptoms which include reduced appetite, emaciation, colour change (darkening), abnormal (whirling) swimming pattern, neurological dysfunction, exophthalmia, swim bladder hyperinflation, floating belly up with inflation of swim bladder, anorexia and gas accumulation and extensive mortality ( Fig. 1) [6,37,38,49,64,77,85,97,102,117,126], (Shetty et al unpublished data). Gross lesions are uncommon, but overinflation of the swimbladder has been observed [37,92].…”

“…To control the infections, it is important to understand the epidemiology and pathogenic mechanisms of the virus. Betanodavirus infection can be influenced by host factors such as age [1,3], environmental factors such as water temperature [106,115,125] and other stress factors such as suboptimal feed, suboptimal water quality, crowding, transport and repeated spawning of broodstock [55,64,84,115]. The virus can be transmitted both horizontally [15,49,71,99] and vertically [2,7,14,49,83,84,92,102,119].…”

“…Therefore, it is very necessary to detect the virus before any clinical signs appear. In addition to microscopic analysis, many sensitive and rapid molecular techniques are available [36,64,89,113,117]. Paying attention to the control measures such as the new generation vaccine strategies is important together with adoption of better management approaches.…”