Macrobrachium rosenbergii nodavirus (MrNV) has been threatening the giant freshwater prawn aquaculture since 1997, causing white tail disease in the prawn species that leads to 100% lethality of the infected postlarvae. Comprehension of the viral infectivity and pathogenesis at molecular biology level has recently resolved the viral capsid protein and evidenced the significant difference in the viral structural protein compared to other nodaviruses that infect fish and insect. Cumulative researches have remarked the proposal to assert MrNV as a member of new genus, gammanodavirus to the Nodaviridae family. The significance of molecular biology in MrNV infection is being highlighted in this current review, revolving the viral life cycle from virus binding and entry into host, virus replication in host cell, to virus assembly and release. The current review also highlights the emerging aptamers technology that is also known as synthetic antibody, its application in disease diagnosis, and its prophylactic and therapeutic properties. The future perspective of synthetic virology technology in understanding viral pathogenesis, as well as its potential in viral vaccine development, is also discussed.
White tail disease in the giant freshwater prawn Macrobrachium rosenbergii causes significant economic losses in shrimp farms and hatcheries and poses a threat to food-security in many developing countries. Outbreaks of Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease (WTD) are associated with up to 100% mortality rates. There are no interventions available to treat or prevent MrNV disease however. Here we show the structure of MrNV virus-like particles (VLPs) produced by recombinant expression of the capsid protein, using cryogenic electron microscopy. Our data show that MrNV VLPs package nucleic acids in a manner reminiscent of other known nodavirus structures. The structure of the capsid however shows striking differences from insect and fish infecting nodaviruses, which have been shown to assemble trimer-clustered T = 3 icosahedral virus particles. MrNV particles have pronounced dimeric blade-shaped spikes extending up to 6 nm from the outer surface of the capsid shell. Our structural analysis supports the assertion that MrNV may belong to a new genus of the Nodaviridae. Moreover, our study provides the first structural view of an important pathogen affecting aquaculture industries across the world.
The giant freshwater prawn/giant river prawn, Macrobrachium rosenbergii is one of the high market value crustaceans cultured worldwide. The intensified aquaculture of the species has led to the outbreak of infectious diseases, prominently, the white tail disease (WTD). It is caused by the infection of Macrobrachium rosenbergii nodavirus (MrNV), which was classified in the family of Nodaviridae. To-date, there are no effective prophylactic and therapeutic agents available against MrNV infection. Vaccination is known to be the most effective prophylactic agent in disease prevention. However, vaccine development against virus infection in crustaceans is equivocal. The feasibility of vaccination in conferring immune protection in crustaceans against infectious diseases is disputable. The argument lies in the fact that crustaceans do not possess adaptive immunity, which is the main immune component that functions to establish immunological memory upon vaccination. Nevertheless, an increasing number of literatures has been documented, which concerns the development of vaccines against infectious diseases in crustaceans. The current review deliberates different approaches in vaccine development against MrNV, which were documented in the past years. It is noteworthy that the live-attenuated MrNV vaccine has not been experimented by far. Thus, the potential of live-attenuated MrNV vaccine in conferring long-term immune protection through the establishment of innate immune memory is currently being discussed.
Progressive research has been recently made in dissecting the molecular biology of Betanodavirus life cycle, the causative pathogen of viral encephalopathy and retinopathy in economic important marine fish species. Establishment of betanodavirus infectious clone allows the manipulation of virus genome for functional genomic study, which elucidates the biological event of the viral life cycle at molecular level. The betanodavirus strategizes its replication by expressing anti-apoptosis/antinecrotic proteins to maintain the cell viability during early infection. Subsequently utilizes and controls the biological machinery of the infected cells for viral genome replication. Towards the late phase of infection, mass production of capsid protein for virion assembly induces the activation of host apoptosis pathway. It eventually leads to the cell lysis and death, which the lysis of cell contributes to the accomplishment of viral shedding that completes a viral life cycle. The recent efforts to dissect the entire betanodavirus life cycle are currently reviewed.
Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant (KDrel) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a KDrel value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a KDrel value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.
The mass mortality of cobia (Rachycentron canadum) within 2-3 days was reported by 3 private farms in Bukit Tambun, Pulau Pinang, in February and March 2007. Only cobia with body weights of 3-4 kg were affected. Most diseased cobia swam on the surface and displayed flashing behaviour. All samples were positive for viral nervous necrosis (VNN) with low to medium levels of infection. Infestations by leeches (Zeylanicobdella arugamensis), body monogeneans (Benedenia sp.) and copepods (Caligus sp.) were also found, but no pathogenic bacteria were isolated. All water quality parameters monitored were within optimal ranges for culturing cobia. The main causes of high mortality in cobia remain unclear during the study. However, we believe that the mass mortality of cobia could be probably due to VNN infection and that the rate of mortality will increase further when cobia are subjected to aquaculture-related stresses (e.g., limited space). Traditional cages with a size of 2 (length) × 2 (width) × 1 m (depth) should only be used for rearing cobia below 1 kg in weight given the species' natural behaviours. In addition, cobia fingerlings should be screened for VNN prior to stocking them in cages.
Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.
Betanodavirus is the causative agent of the viral nervous necrosis (VNN) or viral encephalopathy and retinopathy disease in marine fish. This disease is responsible for most of the mass mortalities that occurred in marine fish hatcheries in Malaysia. The genome of this virus consists of two positive-sense RNA molecules which are the RNA1 and RNA2. The RNA1 molecule contains the RdRp gene which encodes for the RNA-dependent RNA polymerase and the RNA2 molecule contains the Cp gene which encodes for the viral coat protein. In this study, total RNAs were extracted from 32 fish specimens representing the four most cultured marine fish species in Malaysia. The fish specimens were collected from different hatcheries and aquaculture farms in Malaysia. The RNA1 was successfully amplified using three pairs of overlapping PCR primers whereas the RNA2 was amplified using a pair of primers. The nucleotide analysis of RdRp gene revealed that the Betanodavirus in Malaysia were 94.5-99.7% similar to the RGNNV genotype, 79.8-82.1% similar to SJNNV genotype, 81.5-82.4% similar to BFNNV genotype and 79.8-80.7% similar to TPNNV genotype. However, they showed lower similarities to FHV (9.4-14.2%) and BBV (7.2-15.7%), respectively. Similarly, the Cp gene revealed that the viruses showed high nucleotide similarity to RGNNV (95.9-99.8%), SJNNV (72.2-77.4%), BFNNV (80.9-83.5%), TPNNV (77.2-78.1%) and TNV (75.1-76.5%). However, as in the RdRp gene, the coat protein gene was highly dissimilar to FHV (3.0%) and BBV (2.6-4.1%), respectively. Based on the genome analysis, the Betanodavirus infecting cultured marine fish species in Malaysia belong to the RGNNV genotype. However, the phylogenetic analysis of the genes revealed that the viruses can be further divided into nine sub-groups. This has been expected since various marine fish species of different origins are cultured in Malaysia.
Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.
White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.
Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single‑stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.
A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses.
In this article, nine complete genomes of viruses from the genus Alphanodavirus and Betanodavirus (Family Nodaviridae) were comparatively analyzed and the data of their evolutionary origins and relatedness are reported. The nucleotide sequence alignment of the complete genomes from all species and their deduced evolutionary relationships are presented. High sequence similarity within the genus Betanodavirus compared to the genus Alphanodavirus was revealed in multiple sequence alignment of the Nodaviridae genomes. The amino acid sequence similarity for both RNA1 and RNA2 ORF is more conserved in Betanodavirus, compared to Alphanodavirus. The conserved and variable regions within the virus genome that were defined based on the multiple sequence alignments are presented in this dataset.
Discovery of species-specific interaction between the host and virus has drawn the interest of many researchers to study the evolution of the newly emerged virus. Comparative genome analysis provides insights of the virus functional genome evolution and the underlying mechanisms of virus-host interactions. The analysis of nucleotide composition signified the evolution of nodavirus towards host specialization in a host-specific mutation manner. GC-rich genome of betanodavirus was significantly deficient in UpA and UpU dinucleotides composition, whilst the AU-rich genome of gammanodavirus was deficient in CpG dinucleotide. The capsid of MrNV and PvNV of gammanodavirus retains the highest abundance of adenine and uracil at the second codon position, respectively, which were found to be very distinctive from the other genera. ENC-GC3 plot inferred the influence of natural selection and mutational pressure in shaping the evolution of MrNV RdRp and capsid, respectively. Furthermore, CAI/eCAI analysis predicts a comparable adaptability of MrNV in squid, Sepia officinalis than its natural host, Macrobrachium rosenbergii. Thus, further study is warranted to investigate the capacity of MrNV replication in S. officinalis owing to its high codon adaptation index.
Multifunctional nanocarriers displaying specific ligands and simultaneously response to stimuli offer great potentials for targeted and controlled drug delivery. Several synthetic thermally-responsive nanocarriers have been studied extensively for hyperthermia incorporated chemotherapy. However, no information is available on the application of virus-like particle (VLP) in thermally-controlled drug delivery systems. Here, we describe the development of a novel multifunctional nanovehicle based on the VLP of Macrobrachium rosenbergii nodavirus (MrNVLP). Folic acid (FA) was covalently conjugated to lysine residues located on the surface of MrNVLP, while doxorubicin (Dox) was loaded inside the VLP using an infusion method. This thermally-responsive nanovehicle, namely FA-MrNVLP-Dox, released Dox in a sustained manner and the rate of drug release increased in response to a hyperthermia temperature at 43 °C. The FA-MrNVLP-Dox enhanced the delivery of Dox to HT29 cancer cells expressing high level of folate receptor (FR) as compared to CCD841CoN normal cells and HepG2 cancer cells, which express low levels of FR. As a result, FA-MrNVLP-Dox increased the cytotoxicity of Dox on HT29 cells, and decreased the drug's cytotoxicity on CCD841CoN and HepG2 cells. This study demonstrated the potential of FA-MrNVLP-Dox as a thermally-responsive nanovehicle for targeted delivery of Dox to cancer cells rich in FR.
Nodaviruses are small bipartite RNA viruses which belong to the family of Nodaviridae. They are categorized into alpha-nodavirus, which infects insects, and beta-nodavirus, which infects fishes. Another distinct group of nodavirus infects shrimps and prawns, which has been proposed to be categorized as gamma-nodavirus. Our current review focuses mainly on recent studies performed on nodaviruses. Nodavirus can be transmitted vertically and horizontally. Recent outbreaks have been reported in China, Indonesia, Singapore and India, affecting the aquaculture industry. It also decreased mullet stock in the Caspian Sea. Histopathology and transmission electron microscopy (TEM) are used to examine the presence of nodaviruses in infected fishes and prawns. For classification, virus isolation followed by nucleotide sequencing are required. In contrast to partial sequence identification, profiling the whole transcriptome using next generation sequencing (NGS) offers a more comprehensive comparison and characterization of the virus. For rapid diagnosis of nodavirus, assays targeting the viral RNA based on reverse-transcription PCR (RT-PCR) such as microfluidic chips, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and RT-LAMP coupled with lateral flow dipstick (RT-LAMP-LFD) have been developed. Besides viral RNA detections, diagnosis based on immunological assays such as enzyme-linked immunosorbent assay (ELISA), immunodot and Western blotting have also been reported. In addition, immune responses of fish and prawn are also discussed. Overall, in fish, innate immunity, cellular type I interferon immunity and humoral immunity cooperatively prevent nodavirus infections, whereas prawns and shrimps adopt different immune mechanisms against nodavirus infections, through upregulation of superoxide anion, prophenoloxidase, superoxide dismutase (SOD), crustin, peroxinectin, anti-lipopolysaccharides and heat shock proteins (HSP). Potential vaccines for fishes and prawns based on inactivated viruses, recombinant proteins or DNA, either delivered through injection, oral feeding or immersion, are also discussed in detail. Lastly, a comprehensive review on nodavirus virus-like particles (VLPs) is presented. In recent years, studies on prawn nodavirus are mainly focused on Macrobrachium rosenbergii nodavirus (MrNV). Recombinant MrNV VLPs have been produced in prokaryotic and eukaryotic expression systems. Their roles as a nucleic acid delivery vehicle, a platform for vaccine development, a molecular tool for mechanism study and in solving the structures of MrNV are intensively discussed.
The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.