The genus contains four species: Baffin flounder nervous necrosis virus, red spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, and striped jack nervous necrosis virus which is the type species. They were linked with high mortality rates in young fish and were initially described as a Picornavirales -like virus.
They were discovered around the same time in Australia, Norway, France and Japan. The oldest evidence related to the virus, however, dates back to 1984 in Queensland (Australia) where a disease outbreak with the clinical signs and histopathological lesions corresponding to Nov was reported.
EM image and structure of grouper nervous necrosis virus (GNV). Viruses in the genus Betanodavirus are non-enveloped, with icosahedral geometries and T=3 symmetry. The virus -like particle contains 180 subunits of the caps id protein, and each caps id protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold turmeric protrusions.
Genomes are linear, segmented, bipartite, and around 21.4kb in total length. The genome is split in two segments RNA1 and RNA2 that together encode three genes.
Transmission routes are passive diffusion and contact. The disease is more likely to occur in larvae or juveniles but it can also affect adults.
Viral nervous necrosis can have a clinical or sub-clinical presentation. Signs include: abnormal behavior like lethargy, anorexia, spiral swimming; and change in pigmentation.
Microscopical lesions are mostly located in brain, retina and spinal cord where necrosis of the neurons and the presence of round empty spaces called vacuoles are commonly associated with the disease. The most recent common ancestor of the four extant genotypes has been dated to ~1300 CE.
Baffin flounder nervous necrosis virus Red spotted grouper nervous necrosis virus Striped jack nervous necrosis virus Tiger puffer nervous necrosis virus ^ Saul Hammed, AS; Nina we, AS; Sakai, T; Chi, SC; Johnson, KL; CTV Report, Consortium (January 2019).
“CTV Virus Taxonomy Profile: Nodaviridae”. ^ a b More, Koh-Ichiro; Sakai, Toshihiro; Aurora, Kiyokuni; Ariosto, Misdo; Mistake, Each; Kurosawa, IAO (1992).
“Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dented) with nervous necrosis”. “Picorna-like viral particles associated with mass moralities in larval barracuda, Later Calcasieu Bloch”.
“Viral infection (picorna-like virus) associated with mass moralities in hatchery-reared sea-bass (Dicentrarchus Laura) larvae and juveniles”. Rearing and growth of larval and juvenile Barracuda (Later Calcasieu)in Queensland.
Paper presented at: Management of wild and cultured sea bass/barrumundi (Later Calcasieu) (Darwin, Australian Center for International Agricultural Research) ^ Chen, Niece; Yoshimura, Mascot; Gun, Hong-Hsiang; Wang, Tingly; Miami, Yukon; Lin, Chien-Chih; Chuankhayan, Phimonphan; Niagara, At sushi; Chan, Sunday I; Bukhara, Dominate; Chen, Tzong-Yueh; Chen, Chunking (2015). “Crystal Structures of a Pis cine Betanodavirus: Mechanisms of Caps id Assembly and Viral Infection”.
^ Sakai, T.; More, K.; Sugar, T.; Ashoka, T.; Mistake, K.; Yamashina, H. (2009). “Current knowledge of viral nervous necrosis (Van) and its causative betanodaviruses”.
^ Ariosto, Misdo; NATO, Jun; Maryam, Iago; Mira, Gen; Kurosawa, IAO (1996). “Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (Sonny)”.
“Divergence and codon usage bias of Betanodavirus, a neurotrophic pathogen in fish”. Isolation and identification of Singapore grouper rhinovirus Hainan strain (Given) in China | SpringerLink In recent years, with the rapid development of marine farming activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing heavy economic losses.
Singapore grouper rhinovirus (SGI) is one of the most important viruses causing disease in fish. The genome of the isolated virus shared 99.83% nucleotide sequence homology with those of SGI reference strains in the Embank database.
The virus clustered with SGI on an evolutionary tree constructed based on “major caps id protein” (MCP) amino acid sequences, so it was designated ‘Singapore grouper rhinovirus Hainan’ (Given). To evaluate the pathogenic potential of Given in fish, orange-spotted groupers were infected by intrapersonal injection with the virus.
This is the first report of isolation of SGI from naturally infected fish in China, and we show that Given is highly infectious, causing massive deaths in groupers. Huang Oh, Huang Oh, Sun Jr, Han X, Qin Q (2009) Characterization of two grouper Epimetheus Akbar cell lines: application to studies of Singapore grouper rhinovirus (SGI) propagation and virus -host interaction.
Qin QC, Lam TJ, Sin YM, She H, Chang SF, NGO GH, Chen CL (2001) Electron microscopic observations of a marine fish rhinovirus isolated from brown-spotted grouper, Epimetheus Davina. Qin QC, Shi C, Gin KY, Lam TJ (2002) Antigenic characterization of a marine fish rhinovirus from grouper, Epimetheus SPP.
Qin QC, Chang SF, Ngoh-Lim GH, Gibson-Kueh S, Shi C, Lam TJ (2003) Characterization of a novel rotavirus isolated from grouper Epimetheus Davina. Wei J, Zhang X, Gang S, Qin Q (2017) Expression and functional characterization of Trip in orange-spotted grouper (Epimetheus coincides).
The grouper sampling and experiments were approved by the tab of the Animal Care and Use Committee of College of Marine Sciences, South China Agricultural University (HYXY20180501 (Scan)). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Isolation and identification of Singapore grouper rhinovirus Hainan strain (Given) in China. Author: Asia, Muhammad, Jiang, Annie, I, Light, Chen, Winnie, Sun, Linda, Zhao, Juan, Khan Kathak, Muhammad Nair, Tu, Jigging, Lin, Li Source: Virus research 2017 v.227 pp.
245-248 ISSN: 0168-1702 Subject: DNA viruses, Red spotted grouper nervous necrosis virus, alpha-ketoglutaric acid, aquaculture industry, aquatic organisms, carbon, cell viability, financial economics, glutamate, glut amine, humans, nutrients, oxaloacetic acid, Pyrrhic acid, sulfides, carboxylic acid cycle, virus replication Abstract: Glut amine, one of the most important nutrients, plays a vital role in carbon metabolic pathway and has been reported to be required for the replication of several human DNA viruses. In this study, the effects of glut amine on red-spotted grouper nervous necrosis virus (Ronny) replication were investigated.
Picnic pastors surface display of viral caps id proteins was generated in hopes of developing an oral vaccine against red- grouper -nervous-necrosis virus (Ronny) in fish. Fingerlings or juveniles that showed clinical signs of Nov infection were proved by RT-PCR for the appearance of expected length of 198 Buddha and further analysis by DNA sequencing.
The DNA fragment containing AG1 linked to RG-NNVRNA2, 2100 BP in length, was inserted into pPIC9K vector. The extracted cell walls from the yeasts cultured in buffered-methanol-complex medium (BMM) through an induction of 0.5% methanol for 6 days, were investigated for the fusion proteins by western blot.
Further studies are required to optimize fully surface protein expression prior to evaluate the possible use of the constructed recombinant yeast as an oral vaccine against Gonna infection. Epimetheus SPP., or groupers, are in high demand in local and international markets, especially in Singapore and China.
However, with rapid development of marine farming activities, outbreaks of viral nervous necrosis (Van) have affected the grouper aquaculture industry, causing tremendous economic losses and food insecurity . The RNA1 gene in the length of 3.1 KB encodes the 110 KD are replicase for viral replication.
An additional genotype, a turbot betanodavirus strain (TNA) , and several sub-genotypes that appear to be restricted to geographical locations relating to water temperatures have also been proposed . Generally, fish affected by Nov show commonly observed symptoms, including, reduced appetite, dark body and an abnormal swimming pattern.
The disease can be transmitted horizontally in healthy fish commonly through co-habitation in contaminated water and vertically from bloodstock to offspring evidenced, at least in some species . Generally, the mortality rate varies by age and is more pronounced in larvae stages, often higher than 99% .
Oral delivery of vaccines has recently emerged as an attractive alternative to injection in developing countries. They enable mass vaccination at a relatively low cost, easy immunization of fish at all stages, and reduce stress from injection site-pain.
Protection of antigens from deterioration and maintenance of their integrity in the harsh gastrointestinal environment, are two of the key factors for the development of oral vaccines. P. Pastoris can utilize methanol as its sole carbon source under a strong inducible AOX1 promoter to achieve high-level heterozygous protein production .
Antigen determinants that are expressed on the surface of Picnic could be a good candidate for possible oral vaccines. This study described the construction of methylotrophic yeast P. pastors cells displaying Gonna caps id protein linked to the N-terminus of the S. Cartesian GPI-protein, -agglutinin.
In addition, tertiary structure of the fusion protein on the yeast surface was also predicted. Their immunogenicity and efficacy in protecting fish against Gonna by oral delivery might be tested in further studies.
Was cultured at 37C in LB medium containing 100 kg/mL of ampicillin for bacterial colony selection. The wild type S. Cartesian strain was a kind gift from Dr. Banana Limping (Department of Microbiology, Faculty of Sciences, Graph University).
Affected fingerlings or juveniles with the clinical signs of skin darkening, and loss of appetite, anorexia and abnormal swimming behavior were collected during a disease outbreak and sent from Faculty of Veterinary Sciences, Chulalongkorn University. One hundred-milligrams of tissues were homogenized using 1 mL of Trial reagent (Nitrogen, USA) according to manufacturer’s instructions.
The RT-PCR reaction were conducted using VNN-F1 and Inner primer, specific binding to nucleotide(NT) 139 – 336, the conserved region of coat protein of Nov . The CDA product was analyzed with Gene Ruler 100 BP Plus DNA ladder marker (Thermos Scientific, USA) by using 2% agarose gel electrophoresis stained with 10,000-fold diluted SBR Gold (Nitrogen, USA).
The expected length of 198 base pairs (BP) CDA was extracted from agarose gel and purified using Pure Link Quick PCR Purification kit (Nitrogen). Sense strand was sequenced using the specific forward primer (First BASE Laboratories Sinbad, Malaysia) to confirm Ronny genotype based on nucleotide alignment analysis of the open reading frame (Of) of the RNA2 against other Bethesda viruses retrieved from Embank using Dubbed NCBI BLAST program.
GPI-anchored protein AG1 (-agglutinin) was amplified with AG1 Eco RI F and AG1 April R primers in the 50 SL total reaction which was composed of 5 – 10 NG genomic DNA, 1 × GC buffer, 0.2 mm NTPs mix, 0.2 km of each primer and 0.02 U Fusion DNA Polymerase (Thermos Fisher Scientific). The 980 BP of expected PCR products with the addition of Eco RI and April, on 5’ and 3’ terminal end, respectively, were digested with corresponding restriction enzymes and subsequently cloned into the Eco RI and April digested pPIC9K expression vector (Nitrogen, USA).
The identities of plasmids were verified by PCR restriction enzyme digestion, and confirmed by DNA sequencing. Viral RNA's, isolated from affected fish, were used as templates for reverse transcription to produce a single stranded-cDNA by Super Script® III first-strand synthesis system for RT-PCR (Nitrogen, USA).
The first strand of CDA was further transcribed to produce CDA with the Nazi F and FEI R primers corresponding to nucleotides 1 – 23 NT and 993 – 1014 NT of Gonna coat protein, respectively (Table 1) using Fusion DNA Polymerase (Thermos Fisher Scientific). The 1014 BP amplification product containing the partial RNA2 gene of Gonna with the addition of Nazi and FEI restriction site on 5’ and 3’ end, respectively was digested with corresponding restriction enzymes, and finally ligated into Nazi and Eco RI digested pPIC9K-AG-1.
Purified plasmids were verified by PCR using with the alpha factor forward and 3’ AOX1 reverse primer (Nitrogen, USA), specific binding to corresponding region on pPIC9K,and confirmed by DNA sequencing. Fresh colonies of P. pastors yeast strain GS115 (But + His ) (Nitrogen, USA) on Yeast extract pep tone dextrose medium (Yep) plate were cultured in 500 mL PD broth at 30C to OD 600 of 2.4 – 3.0 and collected for the cell pellet by gentrification at 3000× g, 5 min at 4C.
Forty microliters of 1 × 10 10 cells/mL competent cells were mixed with 0.1 kg Bali linearized pPIC9K-RG-NNV RNA2-AG-1 or pPIC9K-AG-1 vector control gently and transferred into 0.2 cm ice-cold cuvette. Immediately added into the mixture and subsequently transferred to 17 × 100 mm glass tube for incubation at 30C, an hour without shaking.
Genomic DNA were extracted and sequenced to determine for correct genomic DNA integration of the expression vectors by PCR, with factor F and 3’AOX1 R primer (Nitrogen, USA) pPIC9K-RG-NNVRNA2-AG1 or pPIC9K-AG1 integrated yeasts were grown at 28C overnight in 50 mL buffered minimal glycerol-complex medium (BMG) to an OD 600 of 2 – 6. After gentrification and resuspension in 1/10 buffered minimal methanol complex medium (BMM) to an OD 600 of 1, the constructs were grown at 28C in a shaking incubator.
After being transferred to polyvinylidene-difluoride membranes using transfer buffer at 50 V overnight, membranes were briefly washed with 1 × buffer followed by incubated with 1000-fold diluted primary antibody (anti-rabbit Nov) for 1.30 h at room temperature followed by incubating with Optimized Hip reagent working dilution (Piece Fast Western Blot Kit, Super Signal West PICO Substrate, Rabbit Thermos Fisher Scientific) for 30 min sat room temperature. Cells were washed in water twice, applied to glass slides, and fixed with 100% acetone for 3 minutes.
Fixed slides were washed in 1 × phosphate buffered saline (PBS) and then incubated with 1:1000 primary antibody (anti-rabbit Nov) at 4C overnight. Fluorescence-labelled cells were then examined under a confocal laser-scanning microscope LSM 800 (Carl Zeiss, Germany).
Total RNA from tissues of affected fingerlings or juveniles showing clinical signs were extracted and analyzed by RT-PCR. A target CDA band of approximately 198 BP on agarose gel electrophoresis was obtained from the amplification of only one affected tissues (Figure 1), and therefore selected to be used for next experimental steps.
RNA2 forms a major Of of caps id protein that is responsible for the host-specificity observed among Betanodavirus isolates. For screening Nov infection, primers were designed based on the DNA sequence of the conserved region among fish noroviruses .
DNA sequencing analysis from amplified CDA showed 92% nucleotide sequence homology to Gonna retrieved from Gen bank database but showed homology to a lesser extent of 81%, 83% and 83% to BFN NV, Sonny and TPN NV, respectively (data not shown). The RT-PCR reactions were conducted using VNN-F1 and Inner, specific binding to nucleotide 139 – 336, the conserved region of coat protein of Nov.
Expected 198 BP of CDA product was analyzed by using 2% agarose gel electrophoresis. In order to construct yeast surface display for antigen, the commercially available pPIC9K expression vector was first used to generate RG-NNVRNA2-AG1 fusion protein (Figure 2).
In this study, however, Sack and Sale linearization of the expression plasmid for insertion at 5’ AOX1 and His 4, respectively were not allowed to produce GS115 But + phenotype. Recombinant P. pastors expression vector for cell surface display of caps id proteins of Gonna.
PPIC9K allows the multiple copy of recombinant genes in P. pastors and presumably brings about a subsequent increase in target protein expression. The level of genetic in resistance can be roughly correlated to vector copy number.
For Must phenotype, these gene replacement strains are selected with the reduced ability to grow on methanol. In order to examine potential expression strains for the foreign gene product, the time-course induction was conducted.
Individual clones and a yeast display vector, pPIC9K-AG1 control were extracted at different time points and analyzed by dot bot analysis. To confirm the identity of spot proteins, crude extracts from selected yeast clones were subsequently separated on 10% SDS-PAGE gel and immunoblot for NNVRNA2.
No protein band was seen in yeast cell extracts from Picnic GS115 transected with the control vector. Thus, the protein band of 56 DA was presumably the non-specific binding of Nov poly clonal antibody in Western blot.
It was noted that differences in expression levels were observed among constructs containing the fusion gene (Figure 3, lane 3 – 6). In this study, all constructs were selected from Yep medium containing 4 mg/mL G418 antibiotic whose level of genetic in resistance could be roughly correlated to the multiple copy number of the desired gene.
This observation suggests that the multiple copy number of genes did not relatively correlate with levels of protein expression in the case of Ronny RNA2. In our case, however, low expression levels of the fusion proteins were detected by western blot analysis.
Cells were extracted and each 20 NG total protein per lane was run on a 10% SDS-PAGE gel. Signal protein bands were detected using 1000-fold diluted primary antibody (anti-rabbit Nov).
Based on molecular weight prediction by NetNGlyc 1.0 program, the coat proteins of Gonna also encompassed 4 N-link glycosylation sites. Furthermore, confocal laser scanning fluorescence microscopy was carried out to visualize the location of the fusion proteins.
Low levels of the fusion protein expression were also observed that agreed with the western blot result. The input amino acid sequence of the fusion protein (685 residues) contained a total of 4% Helix, 15% beta-sheet and 81% loops.
Recombinant P. pastors cells expressing either AG1, vector control (Negative, upper left panel) or the fusion proteins were stained using the antenna antibody. The P domain was the most distal part of the folded coat protein and located at the exterior surface of the caps id.
The P domain is therefore predicted to contain the antigenic determinants of the immunological response of the host. These results confirmed that P domain was not obscured in the protein structure and possible exposed to the immune system.
These results confirmed the correct localization, and exposed conformation of the fusion protein on the cell wall for implication as an oral vaccine. Although the proteins expressed on the yeast surface were readily detected with immunofluorescence, the fluorescence intensity varied among cells.
A methanol feed strategy should be optimized in culture under shake flask conditions to achieve the maximum fusion protein expression as well. In this study, the caps id protein from the red-spotted grouper nervous necrosis virus was successfully expressed and preserved its antigenic integrity on the cell surface of P. pastors.
Optimization to achieve the highest expression of yeast-derived NNVRNA2 on the yeast cell surface is also required before testing, for use as an orally delivered antiviral agent against Gonna infection. This work was financially supported by Agricultural Research Development Agency (Public Organization Grant No.
I am also thankful to Associate Professor Dr. Munchie Payungporn Faculty of Medicine, Chulalongkorn University for experimental equipment, and to John Henderson for kindly editing the manuscript. Intimate, U., Chutney, P., Poomipak, W. and Ararat, N. (2018) Cell Surface Display of Red- Grouper Nervous Necrosis Virus Caps id Protein on Picnic pastors.
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