Search

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans. [ABSTRACT FROM AUTHOR]

Viral infections of teleost fish have great environmental and economic implications in aquaculture. Nervous necrosis virus (NNV) is a pathogen affecting more than 120 different species, causing high mortality and morbidity. Herein, we studied the course of NNV experimental infection of D. labrax, focusing on survivors which indicated viral carrier state. To determine the carrier state of D. labrax head kidney, we performed a gene expression analysis of selected immune-related genes and we profiled its transcriptome 14 days post infection (dpi). All tested genes showed clear differentiations in expression levels while most of them were up-regulated 14 dpi suggesting that their role is not limited in early antiviral responses, but they are also implicated in disease persistence. To gain a better understanding of the fish that survived the acute infection but still maintained a high viral load, we studied the differential expression of 124 up-regulated and 48 down-regulated genes in D. labrax head kidney, at 14 dpi. Concluding, the NNV virus persistent profile was assessed in D. labrax, where immune-related gene modification was intense (14 dpi) and the head kidney transcriptome profile at this time point offered a glimpse into host attempts to control the infection in asymptomatic carriers. [ABSTRACT FROM AUTHOR]

Viral Nervous Necrosis (VNN) and Vibriosis are contagious diseases that can affect groupers from the larval to adult stages and cause significant economic losses for farmers. Sunuk (P. leopardus) grouper is a grouper fish commonly found in NTB waters and has been widely cultivated. This study aims to provide information regarding suspected viral nervous necrosis and vibriosis that may occur in sunuk grouper. This case study was obtained from the fish health inspection process at BKIPM Mataram, involving one of the grouper cultivators in NTB. According to the owner’s explanation, five sunuk grouper were caught by a fisherman and then cultivated in floating net cages. Two sunuk groupers showed injuries in body area and decreased appetite. For several days, two sunuk grouper showed an inactive swimming condition and swam sideways. The result of the physical examination was as follows: fish were swimming limply in an upside-down position and had wounds on various body parts. Virology and bacteriology test confirmed that the sick sunuk grouper was infected with Vibriosis and VNN. Sunuk grouper can be infected with two diseases in this case study, which can occur upon their sick condition from first-day arrival in floating net cages accompanied by stress condition, decreased appetite, that leads to a furher decline in the fish’s health and the immune system not functioning optimally. This condition makes sunuk grouper susceptible to VNN and Vibriosis in this study case. [ABSTRACT FROM AUTHOR]

Cantang grouper (Epinephelus sp.) is one of the Export Oriented commodities which is a mainstay in increasing Indonesian mariculture production. The disease that causes many deaths in cantang grouper is Viral Nervous Necrosis. One of the most effective ways is by giving vaccines to fish. This study aims to develop a recombinant protein Chlorella vulgaris vaccine with the addition of an adjuvant to increase the immunity of cantang grouper infected with VNN. The method used was experimental with a Completely Randomized Design (CRD) consisting of seven treatments and three replications, namely (K+) cantang grouper infected with VNN, (T1) Healthy cantang grouper was given 33 µl of vaccine, (T2) Healthy cantang grouper was given vaccine of 66 µl, (T3) Healthy cantang grouper was given 112 µl of vaccine, (T4) Healthy cantang grouper was given 33 ul of vaccine and then tested against Viral Nervous Necrosis, (T5) Healthy cantang grouper was given 66 ul vaccine and then tested against Viral Nervous Necrosis, (T6) Healthy cantang grouper was given 112 ul vaccine and then tested against Viral Nervous Necrosis. The fish used was 8-10 cm in size and were reared for 56 days. The results showed that the Chlorella vulgaris protein recombinant vaccine with the addition of adjuvant gave a very significant effect on the immunity of cantang grouper infected with VNN with the best dose of 33 µl (T4) seen from the relative protection level (RPS) (75,6%), survival of cantang grouper (78%) after challenge test and a significant increase in antibody titer. [ABSTRACT FROM AUTHOR]

Simple Summary: Redspotted grouper nervous necrosis virus (RGNNV) and its reassortant strains belonging to the genus Betanodavirus represent the most threatening viral pathogens in the Mediterranean aquaculture sector. Due to the sector's ongoing expansion, controlling the spread of viral encephalopathy and retinopathy (VER) caused by betanodaviruses is critical. For this reason, implementing strict hygienic standard procedures primarily based on disinfection could significantly reduce the spread of VER. In this study, two standardized European protocols (BS EN 14675:2015 and BS EN 17111:2018) were applied in order to assess the virucidal activity of a commercial peroxy-acid biocide against the RGNNV and its reassortant RGNNV/SJNNV strain. The disinfectant was effective in reducing the viral titer, but with different results depending on concentrations, strains, and conditions of application. Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the in-field application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure. [ABSTRACT FROM AUTHOR]

Copyright of Jurnal Medik Veterinar is the property of Universitas Airlangga and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Viral nervous necrosis (VNN) is an infectious disease caused by the red-spotted grouper nervous necrosis virus (RGNNV) in European sea bass and is considered a serious concern for the aquaculture industry with fry and juveniles being highly susceptible. To understand the genetic basis for resistance against VNN, a survival phenotype through the challenge test against the RGNNV was recorded in populations from multiple year classes (YC2016 and YC2017). A total of 4,851 individuals from 181 families were tested, and a subset (n∼1,535) belonging to 122 families was genotyped using a ∼57K Affymetrix Axiom array. The survival against the RGNNV showed low to moderate heritability with observed scale estimates of 0.18 and 0.25 obtained using pedigree vs. genomic information, respectively. The genome-wide association analysis showed a strong signal of quantitative trait loci (QTL) at LG12 which explained ∼33% of the genetic variance. The QTL region contained multiple genes (ITPK1 , PLK4 , HSPA4L , REEP1 , CHMP2 , MRPL35 , and SCUBE) with HSPA4L and/or REEP1 genes being highly relevant with a likely effect on host response in managing disease-associated symptoms. The results on the accuracy of predicting breeding values presented 20–43% advantage in accuracy using genomic over pedigree-based information which varied across model types and applied validation schemes. [ABSTRACT FROM AUTHOR]

Viral nervous necrosis (VNN) is an infectious disease caused by the red-spotted grouper nervous necrosis virus (RGNNV) in European sea bass and is considered a serious concern for the aquaculture industry with fry and juveniles being highly susceptible. To understand the genetic basis for resistance against VNN, a survival phenotype through the challenge test against the RGNNV was recorded in populations from multiple year classes (YC2016 and YC2017). A total of 4,851 individuals from 181 families were tested, and a subset (n∼1,535) belonging to 122 families was genotyped using a ∼57K Affymetrix Axiom array. The survival against the RGNNV showed low to moderate heritability with observed scale estimates of 0.18 and 0.25 obtained using pedigree vs. genomic information, respectively. The genome-wide association analysis showed a strong signal of quantitative trait loci (QTL) at LG12 which explained ∼33% of the genetic variance. The QTL region contained multiple genes (ITPK1, PLK4, HSPA4L, REEP1, CHMP2, MRPL35, and SCUBE) with HSPA4L and/or REEP1 genes being highly relevant with a likely effect on host response in managing disease-associated symptoms. The results on the accuracy of predicting breeding values presented 20–43% advantage in accuracy using genomic over pedigree-based information which varied across model types and applied validation schemes.

In December 2019, a mass mortality among cultured Murray cod (Maccullochella peelii peelii) fry occurred on a freshwater farm located at Foshan city of Guangdong province, China. The cumulative mortality was up to 45% within 15 days. The diseased fish showed clinical signs, including abnormal swimming behaviour, loss of appetite and dark body colouration before mass mortality. Samples of brain and retina tissues were collected from affected fish and subjected to reverse transcriptase polymerase chain reaction detection and virus isolation in cell culture. Approximately 430 bp product was detected from the brain and retina tissues and culture supernatant of betanodavirus‐infected SSN‐1 cells. The typical cytopathic effect of betanodavirus infection, which is characterized by vacuolation, was observed in SSN‐1 cells at three days after inoculating with the tissue filtrate of diseased Murry cod fry, and the TCID50 of the infected SSN‐1 cell supernatant was 107.8. Histopathological examinations revealed vacuolation and necrosis in the brain and retina of naturally and experimentally infected Murray cod fry. Electron microscopic observation also showed the aggregation of numerous spherical, non‐enveloped viral particles measuring 22–28 nm in diameter in the cytoplasm of betanodavirus‐infected SSN‐1 cells. Sequence and phylogenetic analysis based on RdRp and Cp genes further indicated that the betanodavirus isolated from Murray cod belonged to the RGNNV genotype. Much higher mortality was obtained in challenged Murray cod fry compared with the controls through immersion challenge. This study is the first report of the natural infection of betanodavirus in freshwater fish in China. [ABSTRACT FROM AUTHOR]

Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the in-field application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure.

Viral nervous necrosis (VNN) is a serious viral disease of several species of farmed and wild fishes. Adult fish are asymptomatic and become carriers of the virus when infected with nervous necrosis virus (NNV) and they transmit the virus to the offspring through eggs. ELISA is ideal for non-lethal screening of adult fish for VNN. Asian seabass (Lates calcarifer) IgM was purified using Protein A affinity column and hybridoma clones secreting monoclonal antibodies (MAb) specific to the heavy chain of IgM was developed. An Indirect ELISA using anti-seabass IgM MAb was developed by optimizing all the reagents. The assay was used to screen adult Asian seabass from grow-out farms in comparison to RT-PCR. The assay was also used to assess the immune response in Asian seabass immunized with inactivated Red-spotted grouper NNV (RGNNV). Seabass IgM on SDS-PAGE analysis revealed three heavy chain bands of size 96, 82 and 76 kDa and a single light chain of size 25 kDa. Out of 18 positive hybridoma clones, two selected clones reacted specifically with the 76 kDa heavy chain band. Out of 28 serum samples of Asian seabass from grow-out farms 26 were positive for NNV antibodies while 22 were positive by RT-PCR. Fish immunized with inactivated RGNNV showed immune response by one week post-immunization, and the peak immune response was observed four weeks post-immunization. The assay developed can be used for non-lethal screening of adult Asian seabass for VNN and to assess the immune response after vaccination.

Background and Aim: Viral nervous necrosis (VNN) is a serious disease of several marine fish species. VNN causes 100% mortality in the larval stages, while lower losses have been reported in juvenile and adult fish. This study aimed to detect the occurrence of VNN while identifying its associated risk factors and the genotypes of its causative agent in a hybrid grouper hatchery in Malaysia. Materials and Methods: A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus X Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors. Results: A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNN-associated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage. Conclusion: This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain.

Viral Nervous Necrosis (VNN) is considered as one of the most important threats for mariculture and aquaculture in the world and up to now, it has been affecting more than 70 fish species. This study was aimed to detect and identify fish betanodavirus, the causative agent of VNN, using nested RT-PCR from brain tissue of Caspian Sea Mullet. Forty samples of subclinical golden grey mullet fish (range: 50-250 gr) were collected from the coastal areas of Caspian Sea in 2016. The target organ (brain) was excised and the reverse transcription (cDNA synthesis) was carried out followed by the RNA extraction. In the present study, detecting of the virus was performed with Nested RT-PCR using two pairs of primers F2/R3 and NF2/NR3 in two steps. After optimization of PCR, all of the forty specimens were negative by the first-step PCR (RT-PCR) while 20 out of 40 grey mullet were positive using Nested RT-PCR (second-step PCR) and a strong band of the expected size (300 bp) was clearly observed. Therefore, it seems that Nested RT-PCR is a method of choice for rapid detection of the low level of infection and in asymptomatic carrier fish. In addition, the result of sequencing revealed that the virus belonged to the RGNNV genotype.

Viral nervous necrosis (VNN), or viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is an acute viral disease of fish, resulting in high mortality globally in cultured marine fish. The pathogen, Betanodavirus belonging to the family Nodaviridae, has been widely documented in both farmed and wild fish, besides in invertebrate species across the habitat barriers. However, cichlid fish, in particular Etroplus species, have a low susceptibility or resistance to this disease. The present study was undertaken to check the susceptibility of a brackishwater cichlid, orange chromide (Etroplus maculatus) through intramuscular injection and immersion challenge by NNV (RGNNV genotype). Intramuscular injection (10 μl) with NNV (105 viral particles ml−1) resulted in cumulative mortality of 50% at 15 days of post-infection (dpi), while immersion challenge in NNV spiked sea water (105 TCID50 ml−1) resulting in a mortality of 25% at 19 dpi. Unlike many marine fish which showed 100% mortality within 3–5 days, E. maculatus challenged through both the routes exhibited severe clinical symptoms for 2–3 weeks followed by a recovery stage and moderate level of mortality. Reverse transcription polymerase chain reaction (RT-PCR) tests on live fish sampled during the course of the experiment and the dead fish exhibiting symptoms in both challenge groups tested NNV-positive. Histological examination of fish in challenged groups showed VNN-specific lesions characterized by vacuolations in the brain and eye with more severe lesions in the injected group than the immersion group. In survived fish, no clinical sign was observed, but NNV could be detected by RT-PCR with moderate to severe vacuolations in the brain and eyes until 30 dpi. Despite the virus entering the body and triggering typical symptoms of VNN, the fish appeared to exhibit moderate resistance with a survival of 50–75%. The results indicate that E. maculatus could serve as a brackishwater fish model to study host-pathogen interaction and development of resistance for potential application in NNV-vaccine studies. [ABSTRACT FROM AUTHOR]

Nervous necrosis virus (NNV) has been responsible for mass mortalities in the aquaculture industry worldwide, with great economic and environmental impact. The present review aims to summarize the current knowledge of gene expression responses to nervous necrosis virus infection in different fish species based on transcriptomic analysis data. Four electronic databases, including PubMed, Web of Science, and SCOPUS were searched, and more than 500 publications on the subject were identified. Following the application of the appropriate testing, a total of 24 articles proved eligible for this review. NNV infection of different host species, in different developmental stages and tissues, presented in the eligible publications, are described in detail, revealing and highlighting genes and pathways that are most affected by the viral infection. Those transcriptome studies of NNV infected fish are oriented in elucidating the roles of genes/biomarkers for functions of special interest, depending on each study’s specific emphasis. This review presents a first attempt to provide an overview of universal host reaction mechanisms to viral infections, which will provide us with new perspectives to overcome NNV infection to build healthier and sustainable aquaculture systems.

Aim: The aim of the study was to determine the prevalence and risk factors of viral nervous necrosis (VNN) disease in tiger grouper cultured in the floating net cage in Bintan district, Indonesia. Materials and Methods: Sampling used multiple stages with ponds as interest units. The selection conducted by systematic random sampling from the entire net cage in Bintan district. The fish samples were selected based on the appearance of clinical signs of infected fish. The risk factors investigated in this study included net cage, technical, and sample fish information, culture, water quality, and feed management. A total of 195 fishes pooled to the 39 net cages and tested using the nested polymerase chain reaction technique to determine the VNN status. The brain and eye processed for histopathology. Results: The prevalence rate of VNN on the net cage was 38% (15/39). The risk factors affecting VNN using bivariate analysis was cleaning the net (χ2=9.80; p=0.002), replacement of net cage (χ2=5.20; p=0.0226), and floating net cage technicians knowledge (χ2=4.13; p=0.042). The variables of positive risk factors affecting VNN by multivariate analysis were the juvenile source and the level of mid-weather changes (technician experience and dissolved oxygen [DO]). Seven variables associated to the VNN outbreak have detected. The positive multiplier factors were the source of juveniles, mid weather changes, technician experience, and the DO, while the negative factors were salinity, mixed feed, and the low weather changes. Histopathologically, the grouper fish showed the brain, eye, and muscle vacuolization and kidney necrosis. Conclusion: It proves that the Bintan waters contaminated by VNN had a prevalence rate of 38% from the total sample based on bivariate method, net cleaning, net replacement, and knowledge on the VNN outbreak. Natural infection of VNN in grouper leads to vacuolization of the brain, the eye, and muscles nearby the eye as well the kidney necrosis.

Viral nervous necrosis (VNN) caused by the nervous necrosis virus (NNV) affects a broad range of primarily marine fish species, with mass mortality rates often seen among larvae and juveniles. Its genetic diversification may hinder the effective implementation of preventive measures such as vaccines. The present study describes different inactivation procedures for developing an inactivated vaccine against a new NNV isolate confirmed to possess deadly effects upon the European seabass (Dicentrarchus labrax), an important Mediterranean farmed fish species that is highly susceptible to this disease. First, an NNV isolate from seabass adults diagnosed with VNN was rescued and the sequences of its two genome segments (RNA1 and RNA2) were classified into the red-spotted grouper NNV (RGNNV) genotype, closely clustering to the highly pathogenic 283.2009 isolate. The testing of different inactivation procedures revealed that the virus particles of this isolate showed a marked resistance to heat (for at least 60 °C for 120 min with and without 1% BSA) but that they were fully inactivated by 3 mJ/cm2 UV-C irradiation and 24 h 0.2% formalin treatment, which stood out as promising NNV-inactivation procedures for potential vaccine candidates. Therefore, these procedures are feasible, effective, and rapid response strategies for VNN control in aquaculture.

Two disease outbreaks were encountered in cage cultured cobia fingerlings with the mortality of 36.80% and 83.70% during summer month at Palk Bay and Gulf of Mannar, respectively. The mortality started with the appearance of clinical signs like off feed, erratic swimming behavior with circling movement, surfacing, and acute death. The gross lesions observed were corneal opacity, subsequent congestion, exophthalmosis, abdominal distention with ascites, severe congestion at the caudal lobe of the liver, and moderate congestion of the brain. Histopathologically, moderate vacuolation and spongiosis were observed in brain. The viral nervous necrosis was confirmed by RT-PCR from the brain samples collected from the affected fish. Addition to this viral disease, Vibrio alginolyticus (KX989540) was isolated from Palk Bay cage cultured fingerlings and Pseudomonas aeuruginosa (KX860116) from Gulf of Mannar. The sea surface water temperature during the disease outbreak was significantly (P < 0.05) higher. In conclusion, two outbreaks were due to co-infection by VNN and bacteria. The higher temperature during the months of April and May (33.2 ± 0.25 °C 34.1 ± 0.25 °C), increased stocking density, and occlusion of barnacles and clams at the cage inner net were attributed as main stress factors that might have induced the infection resulted in mass mortalities of cobia fingerlings. • In the two epidemics, 36% of the deaths were attributable to VNN combined with V. alginolyticus infection and 83% to VNN and co-infection with P. aeuruginosa. • The main stress variables that contribute to viral infection with bacterial co-infection have been found to be higher temperature, higher stocking densities, and barnacle obstruction at the cage inner net. • These findings provide insight into the predisposing elements responsible for the onset of infection in cage-cultured fish, allowing improved managemental approaches to control and prevent infection. [ABSTRACT FROM AUTHOR]

The rapidly increasing Mediterranean aquaculture production of European sea bass is compromised by outbreaks of viral nervous necrosis, which can be recurrent and detrimental. In this study, we evaluated the duration of protection and immune response in sea bass given a single dose of a virus-like particle (VLP)-based vaccine. Examinations included experimental challenge with nervous necrosis virus (NNV), serological assays for NNV-specific antibody reactivity, and immune gene expression analysis. VLP-vaccinated fish showed high and superior survival in challenge both 3 and 7.5 months (1800 and 4500 dd) post-vaccination (RPS 87 and 88, OR (surviving) = 16.5 and 31.5, respectively, p < 0.01). Although not providing sterile immunity, VLP vaccination seemed to control the viral infection, as indicated by low prevalence of virus in the VLP-vaccinated survivors. High titers of neutralizing and specific antibodies were produced in VLP-vaccinated fish and persisted for at least ~9 months post-vaccination as well as after challenge. However, failure of immune sera to protect recipient fish in a passive immunization trial suggested that other immune mechanisms were important for protection. Accordingly, gene expression analysis revealed that VLP-vaccination induced a mechanistically broad immune response including upregulation of both innate and adaptive humoral and cellular components (mx, isg12, mhc I, mhc II, igm, and igt). No clinical side effects of the VLP vaccination at either tissue or performance levels were observed. The results altogether suggested the VLP-based vaccine to be suitable for clinical testing under farming conditions.

Background and Aim: Viral nervous necrosis (VNN) is a serious disease of several marine fish species. VNN causes 100% mortality in the larval stages, while lower losses have been reported in juvenile and adult fish. This study aimed to detect the occurrence of VNN while identifying its associated risk factors and the genotypes of its causative agent in a hybrid grouper hatchery in Malaysia. Materials and Methods: A batch of newly hatched hybrid grouper fry (Epinephelus fuscoguttatus × Epinephelus lanceolatus) were followed from the larval stage to market size. Samples of the hybrid groupers, water, live feed, and artificial fish pellets were collected periodically from day 0 to 180 in the hybrid grouper hatchery. Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR amplifications were carried out on VNN-related sequences. The phylogenetic tree including the sampled causative agent of VNN was inferred from the coat protein genes from all known Betanodavirus species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson's correlation coefficient values were calculated to determine the strength of the correlation between the presence of VNN in hybrid grouper samples and its associated risk factors. Results: A total of 113 out of 146 pooled and individual samples, including hybrid grouper, water, and artificial fish pellet samples, demonstrated positive results in tests for the presence of VNN-associated viruses. The clinical signs of infection observed in the samples included darkened skin, deformation of the backbone, abdominal distension, skin lesions, and fin erosion. VNN was present throughout the life stages of the hybrid groupers, with the first detection occurring at day 10. VNNassociated risk factors included water temperature, dissolved oxygen content, salinity, ammonia level, fish size (adults more at risk than younger stages), and life stage (age). Detection of VNN-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 100% to each other, striped jack nervous necrosis virus (SJNNV), and the reassortant strain red-spotted grouper NNV/SJNNV (RGNNV/SJNNV) isolate 430.2004 (GenBank accession number JN189932.1) (n=26). The phylogenetic analysis showed that quasispecies was present in each VNN-causing virus-positive sample, which differed based on the type of sample and life stage. Conclusion: This study was the first to confirm the existence of a reassortant strain (RGNNV/SJNNV) in hybrid groupers from Malaysia and Southeast Asia. However, the association between the mode of transmission and the risk factors of this virus needs to be investigated further to understand the evolution and potential new host species of the reassortant strain. [ABSTRACT FROM AUTHOR]

We determined the complete genomic RNA sequence of a new type of betanodavirus Korea shellfish nervous necrosis virus (KSNNV) isolated from shellfish. Compared with other isolates representing four genotypes of betanodaviruses, the identity of the whole nucleotide sequence of the virus was in the range of 76%–83% with the presence of specific genetic motifs and formed a separate new branch in the phylogenetic analysis. In pathogenic analysis by immersion method, KSNNV‐KOR1 shows 100% cumulative mortality like SFRG10/2012BGGa1 (RGNNV) in newly hatched sevenband grouper and mandarin fish, which is clearly different from those found in negative control groups. There were no significant differences in increasing rates of mortality and viral intra‐tissue concentration of larval fishes infected with KSNNV‐KOR1 at both 20 and 25°C water temperature. Histopathological examination of each fish species in the moribund stage revealed the presence of clear vacuoles in both brain and retinal tissues similar to typical histopathology features of RGNNV. In the present study, we first report a new betanodavirus from shellfish as the aetiological agent of viral nervous necrosis disease in fish with complete genomic nucleotide sequence and pathogenic analysis. [ABSTRACT FROM AUTHOR]

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture. [ABSTRACT FROM AUTHOR]

Summary: Betanodaviruses cause the disease viral nervous necrosis (VNN) in finfish. Using a novel approach with two consecutive PCRs, detection semi‐nested two‐step RT‐PCR (DSN‐2 RT‐PCR) and discriminative multiplex two‐step RT‐PCR (DMT‐2 RT‐PCR), we have identified the presence of a new type of betanodavirus in shellfish and called it Korean shellfish nervous necrosis virus (KSNNV). Partial nucleotide sequences of the T4 region in RNA2 fragment of KSNNVs were 73%–75% homologous to those of other reported genotypes and formed a new cluster of betanodavirus in phylogenetic tree analysis. Successful isolation of KSNNV was achieved in two of six shellfish samples containing high concentrations of virus using the blind passage method, and the typical shapes of betanodavirus were confirmed in KSNNV‐KOR1 by electron microscopy. In the experimental infection test, seven of 14 fish species showed susceptibility to KSNNV‐KOR1 isolate but without clinical signs or death. Although the range of susceptible host species was not significantly different from the RGNNV type, the concentration of KSNNV in the brain of infected fish (102–105 copies/mg brain) was much lower compared to that found in sevenband grouper (Epinephelus septemfasciatus Thunberg) sampled in the moribund stage with RGNNV infection (106–107 copies/mg brain). However, histopathological analyses showed the presence of multiple vacuoles in brains of all KSNNV‐infected fish at 14 days postinjection. In detection test, as a single or multiple type with the other genotype(s) (RGNNV or BFNNV), the prevalence of KSNNV was 8.4% and 8.7% in domestic (62 of 741 samples) and Chinese samples (12 of 138 samples), respectively, but not in finfish. We propose that KSNNVs obtained from shellfish be classified into a separate and new genotype of betanodavirus. [ABSTRACT FROM AUTHOR]

The purpose of study is to determine the type of plankton found in cultivation pond of nile tilapia infected by VNN. The method is to identify the plankton in the water pond and testing the VNN that infects nile tilapia related to plankton eaten using Polymerase Chain Reaction (PCR). VNN identification results on the nile tilapia digestive tract stomach and intestines as well as plankton found in the stomach and intestines indicate VNN positively. Plankton found in the digestive tract of fish is division of Chlorophyta, Bacillariophyta, phylum Rotifera and Crustacea. It indicates that nile tilapia infected by VNN both on the gastrointestinal tract organs, stomatch or intestines and plankton were found with positive VNN digestive duct, while plankton found in water pond is VNN negative. The VNN infection depends on the nature of specifications VNN on nile tilapia and the plankton in the nile tilapia digestive duct.

Betanodavirus are the causative agents of viral nervous necrosis (VNN) in cultured marine fish, as Sea bass (Lates calcarifer). The aims of current study toward in establishing the rapid method to detect the presence of Betanodavirus in Sea bass. A total of 30 death aquarium Sea bass, which were collected from Ben Tre province, Vung Tau province and positive control which confirmed within viral nervous necrosis, were enrolled into present study. Initially, we were successful in primers designed, which were also confirmed the specificity to Betanodavirus and sensitivity which reached at least 103 copies/ml. Finally, we successfully adopted rapid detection of Betanodavirus presence based on RT-PCR. As the results, 3 of 30 cases were positive to Betanodavirus. Then, the results were confirmed by sequencing and the similarity was showed. Therefore, in further study, it will be continuous in detecting the presence of Betanodavirus in various kinds of fishery samples.

Aim: The aim of the study was to determine the prevalence and risk factors of viral nervous necrosis (VNN) disease in tiger grouper cultured in the floating net cage in Bintan district, Indonesia. Materials and Methods: Sampling used multiple stages with ponds as interest units. The selection conducted by systematic random sampling from the entire net cage in Bintan district. The fish samples were selected based on the appearance of clinical signs of infected fish. The risk factors investigated in this study included net cage, technical, and sample fish information, culture, water quality, and feed management. A total of 195 fishes pooled to the 39 net cages and tested using the nested polymerase chain reaction technique to determine the VNN status. The brain and eye processed for histopathology. Results: The prevalence rate of VNN on the net cage was 38% (15/39). The risk factors affecting VNN using bivariate analysis was cleaning the net (χ²=9.80; p=0.002), replacement of net cage (χ²=5.20; p=0.0226), and floating net cage technicians knowledge (χ²=4.13; p=0.042). The variables of positive risk factors affecting VNN by multivariate analysis were the juvenile source and the level of mid-weather changes (technician experience and dissolved oxygen [DO]). Seven variables associated to the VNN outbreak have detected. The positive multiplier factors were the source of juveniles, mid weather changes, technician experience, and the DO, while the negative factors were salinity, mixed feed, and the low weather changes. Histopathologically, the grouper fish showed the brain, eye, and muscle vacuolization and kidney necrosis. Conclusion: It proves that the Bintan waters contaminated by VNN had a prevalence rate of 38% from the total sample based on bivariate method, net cleaning, net replacement, and knowledge on the VNN outbreak. Natural infection of VNN in grouper leads to vacuolization of the brain, the eye, and muscles nearby the eye as well the kidney necrosis. [ABSTRACT FROM AUTHOR]

Outbreaks of viral nervous necrosis (VNN) in Asian sea bass ( Lates calcarifer ) at the larval stages via vertical transmission of nervous necrosis virus (NNV) from asymptomatic broodfish remain as a major deterrent during seed production. A five-year study was conducted to produce NNV-specific-free sea bass broodfish reared in land-based tanks through an annual immunization regimen with the formalin-inactivated NNV. We primarily immunized (intraperitoneal injection) sea bass juveniles (5 g) and monitored the neutralizing antibody (Nab) titers in the sera of these fish at scheduled intervals post-immunization. Nab titers in the sera of immunized fish peaked at Month 2 (titer: 1:4480 ± 1185) but thereafter gradually declined and significantly dropped (1:260 ± 83) at Month 12 post-primary immunization. Booster immunization of these fish at Month 12 post-immunization led to abrupt increases in Nab titers in booster immunized (B-Im) fish at Month 1 (1:12800 ± 6704) but thereafter declined and dropped at Month 12 (1:480 ± 165) post-booster immunization. The annual booster injections with the inactivated vaccine or L-15 (Unimmunized [U-Im]) were consecutively conducted for 4 years until the fish became sexually mature. Mature fish from both groups were successively induced to spawn twice (1-month interval) via intramuscular injection with luteinizing hormone-releasing hormone analogue (LHRH-a; 100 µg/kg BW). NNV was not detected by RT-PCR in oocytes and milts, and spawned eggs of B-Im fish. In contrast, oocytes and milts, and spawned eggs of U-Im fish were NNV positive. Spawned eggs of B-Im broodfish exhibited Nab titers ranging from 1:192 ± 34 to 1:240 while such was not detected (<1:40) in eggs of U-Im fish. Taken together, current data clearly demonstrate that annual immunization regimen with inactivated NNV vaccine is a pragmatic approach for sustaining immunocompetent sea bass broodfish reared in land-based tanks and circumvent the risk of vertical transmission of NNV from asymptomatic broodfish to their offspring under stress of repetitive spawning. [ABSTRACT FROM AUTHOR]

Betanodaviruses are the causative agents of the disease known as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in a variety of marine and freshwater fish species. The aim of this study was to demonstrate experimental infection of an isolate of betanodavirus (RGNNV genotype) in freshwater fish, Gambusia affinis, for elucidation of transmission mechanism and potential use as a laboratory model. Morbidity and mortality rate was significantly higher by injection route of infection as compared to immersion by bath and resembled the natural infection of juvenile marine fish. The fish in disease affected group showed severe neurological disorders accompanied by extensive vacuolar degeneration and mild to moderate neuronal necrosis of the brain in comparison to control. Amplification of ~ 427 bp product in the variable region of the coat protein gene of betanodavirus was achieved by RT-PCR with 100% sequence homology to RGNNV genotype. [ABSTRACT FROM AUTHOR]

Viral nervous necrosis is a serious viral disease of marine and brackishwater finfish hatcheries. The red-spotted grouper nervous necrosis virus (RGNNV) is the most prevalent genotype among fishes in tropical and temperate regions. RGNNV was propagated in SSN-1 cell line and inactivated using BEI. The capsid protein of RGNNV was cloned and expressed in Escherichia coli and purified. Inactivated and recombinant VNN vaccines were produced by emulsifying the antigen with commercial adjuvant and administered to Asian seabass, Lates calcarifer , fingerlings and broodstock intraperitoneally. The immune response was assessed at regular intervals by indirect ELISA. The maternal antibodies in one-day post-hatch larvae obtained from the vaccinated and control broodstock were assessed by indirect ELISA. The larvae were subjected to challenge with virulent RGNNV and the relative per cent survival (RPS) was estimated. The efficacy of the vaccine was also assessed under field conditions. The recombinant vaccine produced significantly higher specific antibody levels than the inactivated vaccine in fingerlings and broodstock and the immune response was dose-dependent. Recombinant vaccine at 50 and 100 μg/fish produced significantly higher antibody levels and immune response could be observed as early as one-week post vaccination. Vaccinated broodstock produced larvae with maternal antibodies and had higher RPS when challenged at 18 days post-hatch. The vaccine produced a significant immune response even under field conditions. The vaccine can be used to vaccinate broodstock to reduce loss due to VNN in early larval stages. • Recombinant vaccine produced better immune response than inactivated vaccine. • Recombinant vaccine dose of 50 μg/fish is optimum for vaccination of Asian seabass fingerlings. • Vaccination of brooders resulted in transfer of maternal antibodies to offspring. • Asian seabass larvae obtained from vaccinated stocks had higher RPS upon challenge. [ABSTRACT FROM AUTHOR]

Infectious disease is a major challenge in aquaculture and poses a constraint for the development of farming of new species. In 2017, Siberian sturgeon (Acipenser baerii) juveniles were imported from Italy to a Swedish farm. Transport conditions were suboptimal. Thirty percent died during transport and within the first days after arrival. Ten days after arrival, mortalities started to occur again, which prompted initiation of an investigation into the mortalities. Diseased fish were transported live to the National Veterinary Institute (SVA) for necropsy and further analysis. Pathological and histopathological investigation was conducted. Virology was performed on gills and internal organs by cell culture isolation and using specific PCR protocols against nervous necrosis virus (NNV) and Acipenser iridovirus European (AcIV-E). The juveniles displayed neurological signs such as lethargy, inability to maintain an upright position, and erratic swimming. Body condition was below normal, and gills were pale. One fish had petechial hemorrhages on the abdomen and the snout. Two specimens had intestinal hyperemia. Ventricles were air-filled, and swim bladders were deflated. Viral cell cultures gave negative results, but PCR analysis of gills and internal organs detected the presence of AcIV-E. We conclude that AcIV-E was associated with disease and high mortality in the sturgeon juveniles, and stress probably aggravated the course of the infection.

Three of the most important diseases of Mediterranean intensive European sea bass farming are, viral nervous necrosis (VNN) caused by the red grouper nervous necrosis virus (RGNNV) genotype of b-nodavirus, photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp) and vibriosis caused mainly by the O1 serotype of Vibrio anguillarum (VaO1). Prevention against these diseases is performed through vaccination with a monovalent vaccine against the viral disease and, usually, with bivalent vaccines against the bacterial diseases. However, it is very difficult to program two vaccinations during the same season for the same fish stock and producers are forced to either vaccinate for the viral or the bacterial diseases or to perform double vaccination with both vaccines, without any prior knowledge on any interactions that may occur due to the plethora of antigens (Ag) injected. Ideally, therefore, a trivalent vaccine should be developed against all three diseases. The objective of this work was to analyse the immune response of sea bass against combinations of Ags from all three pathogens, namely viral particles, Phdp whole cells (WC), lipopolysaccharide (LPS), capsular polysaccharide (CPS) and extracellular products (ECPs) and VaO1 WC and ECPs in respect to the identification of any phenomena of immunodominance/immunosuppression between Ags with a view to select candidate Ags for inclusion in a trivalent vaccine formulation. Eight triplicate groups of fish were immunized with different combinations of the aforementioned Ags and another triplicate group served as negative control. Blood serum was isolated at various time-points post-immunization for the measurement of specific antibodies against each Ag and, in addition, leucocytes were isolated at day 29 post-immunization for analysis of various cellular activities. Results indicated that best levels of specific a-NNV virus antibodies (Abs) were produced when VaO1 ECPs were not included in the Ag combinations, in contrast to the leucocytes proliferation assay where best stimulation against NNV Ags was measured when VaO1 ECPs were present in Ag combinations. VaO1 ECPs apparently is a strong immunogen for both humoral and cellular responses but suppresses immunological reactions against the other Ags.VaO1 WC, Phdp LPS and ECPs raised good humoral immune responses in the groups with best responses against VNN Ags, but only VaO1 WC and Phdp ECPs provided good stimulation of leucocytes, with Phdp WC and CPS effecting either similar stimulation with untrained leucocytes (control groups) or down-stimulation. Results are discussed with a view to select Ags from all three pathogens for inclusion in trivalent vaccine against all three pathogens. [ABSTRACT FROM AUTHOR]

The present study was conducted on 428 moribund mullet fish samples to isolate and identify the causative agent of a mysterious acute mortality which recently occurred in wild mullets in Iranian waters of Caspian Sea, suspected to be due to viral nervous necrosis (VNN) disease. Disease investigation was carried out employing various diagnostic procedures such as virology, bacteriology, parasitology, haematology, histopathology, IFAT, IHC and nested RT-PCR. Brain and eye samples of affected fishes were collected in sterile conditions and then kept at −80 °C for cell culture isolation and nested RT-PCR detection of the causative agent. Other tissue samples were also collected and fixed for histopathology, IHC and EM examinations. CPE was observed in cell cultures at 6 days after inoculation. Nine samples were found positive with virological assay. Nested RT-PCR, performed on suspected tissues and CPE positive samples, showed that about 21 tissue samples and all the CPE positive samples were positive for VNN virus (VNNV). IFAT was selected as a confirmatory method for detecting the presence of Betanodavirus antigen, cell culture isolation results and nested RT-PCR findings. Moreover, VNNV particles with 25–30 nm in diameter were also visualized in the infected brain and retina. In pathogenicity studies, guppy fishes bathed in VNNV-infected tissue culture (10 −4 TCID 50 ) showed clinical signs similar to naturally infected mullet after 15 days post infection (dpi), with mortality rates reaching up to 100% at 30 dpi. Affected organ samples as examined by cell culture isolation, IFAT, IHC and histopathology, revealed the presence of VNNV in the guppy fishes. In conclusion, it was confirmed that VNNV was the main causative agent for the disease outbreak in mullet fish in the Caspian Sea, and this is such first official report of VNN disease from Iran. [ABSTRACT FROM AUTHOR]

Betanodavirus is a small bipartite RNA virus of global economical significance that can cause severe neurological disorders to an increasing number of marine fish species. Herein, to further the understanding of the evolution of betanodavirus, Bayesian coalescent analyses were conducted to the time-stamped entire coding sequences of their RNA polymerase and coat protein genes. Similar moderate nucleotide substitution rates were then estimated for the two genes. According to age calculations, the divergence of the two genes into the four genotypes initiated nearly simultaneously at ∼700 years ago, despite the different scenarios, whereas the seven analyzed chimeric isolates might be the outcomes of a single genetic reassortment event taking place in the early 1980s in Southern Europe. Furthermore, codon usage bias analyses indicated that each gene had influences in addition to mutational bias and codon choice of betanodavirus was not completely complied with that of fish host. [ABSTRACT FROM AUTHOR]

Liza klunzingeri is an important species in the Persian Gulf and Oman Sea. A large number of L. klunzingeri suffered acute mortality in the Persian Gulf and Oman Sea coasts. The infected fish showed some clinical signs such as abnormal and whirling swimming, changes in pigmentation, swelling of swim bladder, and belly-up position. The affected fish showed hemorrhage in lateral and abdominal cavities. Also, hemorrhages were seen in the operculum, and thoracic and caudal fins. Abdominal cavity had no abnormality. For histological examination, the samples were fixed in Bouin's fixative. After tissue processing, 5-μm sections were stained with hematoxylin and eosin. Microscopic observation of spleen, liver, intestine, gills, and kidney tissue showed no morphological and histopathological changes. However, necrosis and vacuolation were seen in both brain and retinal tissues. Lesions were severe and widespread in the gray matter compared to white matter. The current report is the first description and account of an acute mortality with viral nervous necrosis similar clinical signs and histopathology in the Persian Gulf and Oman Sea involving L. klunzingeri. [ABSTRACT FROM AUTHOR]

The article discusses the betanodavirus-associated mortalities of adult wild groupers epinephelus costae (Steindachner) and epinephelus marginatus (Lowe). Topics include marine fish in the Mediterrreanean Sea, deoxyribonucleotide triphosphates (dNTPs), and phylogenetic analysis. Also provided is information regarding domestic and industrial pollution, dead fish, and ectoparasites.

The article presents a study on the elevation of temperature and crowding in laboratory model zebra fish with viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) caused by betanodavirus. The study has experimented two batches of subclinically infected model zebra fish which were randomly sampled. The results state the presence of the virus without manifestation and cumulative mortality.

Frequent mortality was observed in the larval rearing facility of marine clownfish, Amphiprion sebae Bleeker. Mortality resulted in 80% loss of larval stock. Moribund larvae showed clinical signs typical of viral nervous necrosis, such as uncoordinated, corkscrew-like swimming behaviour, hypersensitivity to stimuli, darkening of body and assembly into large groups, similar to bunches of grapes. The aetiology of the disease was confirmed by gross observation of clinical signs, histopathology and molecular diagnosis. Histological studies revealed severe vacuolation in the brain and in the bipolar and ganglion layers of the eye. Molecular diagnosis by reverse transcription-polymerase chain reaction (RT- PCR) specific to piscine nodavirus yielded a positive result. The partial nucleotide sequences of the PCR-amplified fragment were 97-98% similar to other betanodavirus isolates reported globally and more closely aligned with red-spotted grouper nervous necrosis virus ( RGNNV). This is the first report of susceptibility of clownfish, A. sebae, to betanodavirus and the presence of the RGNNV in India. [ABSTRACT FROM AUTHOR]