Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?

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.

Noda-Like RNA Viruses Infecting Caenorhabditis Nematodes: Sympatry, Diversity, and Reassortment.

Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative, Caenorhabditis briggsae Here, we report on a collection of more than 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Melník virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of coinfection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative, C. briggsae, and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Melník virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.

ICTV Virus Taxonomy Profile: Nodaviridae.

The family Nodaviridae includes two genera, Alphanodavirus and Betanodavirus. The family name derives from the Japanese village of Nodamura where Nodamura virus was first isolated from Culex tritaeniorhynchus mosquitoes. Virions are non-enveloped and spherical in shape with icosahedral symmetry (T=3) and diameters ranging from 25 to 33 nm. The genome consists of two molecules of single-stranded positive-sense RNA: RNA1 and RNA2. The virion capsid consists of 180 protein subunits arranged on a T=3 surface lattice. Alphanodaviruses infect insects, whereas betanodaviruses are pathogens of fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Nodaviridae, which is available at www.ictv.global/report/nodaviridae.

Infection of covert mortality nodavirus in Japanese flounder reveals host jump of the emerging alphanodavirus.

Interspecies transmission of viruses, where a pathogen crosses species barriers and jumps from its original host into a novel species, has been receiving increasing attention. Viral covert mortality disease, caused by covert mortality nodavirus (CMNV), is an emerging disease that has recently had a substantial impact on shrimp aquaculture in Southeast Asia and Latin America. While investigating the host range of CMNV, we found that this virus is also capable of infecting populations of the farmed Japanese flounder Paralichthys olivaceus, a vertebrate host. The infected fish were being raised in aquaculture facilities that were also producing marine shrimp. Through RT-nPCR, targeting the RNA-dependent RNA polymerase (RdRp) gene of CMNV, we found that 29 % of the fish sampled were positive. The amplicons were sequenced and aligned to the RdRp gene of shrimp CMNV and were found to have 98 % identity. Histopathological examination indicated that CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain, as well as extensive necrosis of cardiac muscle. In situ hybridization showed positive reactions in tissues of the eye, brain, heart, liver, spleen and kidney of infected fish. Transmission electron microscopy showed the presence of CMNV-like particles in all of the above-mentioned tissues, except for brain. The novel finding of a shrimp alphanodavirus that can also infect farmed P. olivaceus indicates that this virus is capable of naturally crossing the species barrier and infecting another vertebrate. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Lack of in vivo cross-protection of two different betanodavirus species RGNNV and SJNNV in European sea bass Dicentrachus labrax.

Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described. Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro. In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV. Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus. These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.

Occurrence and molecular characterization of betanodaviruses in fish and invertebrates of the Greek territorial waters.

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.

Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs.

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red-spotted grouper nervous necrosis virus genotype in shellfish.

Using two serially executed PCRs, the discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR) following the detection seminested two-step RT-PCR (DSN-2 RT-PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable in vitro. These results are the first report of high level prevalence of in vitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.

Genotype-specific real-time PCR combined with high-resolution melting analysis for rapid identification of red-spotted grouper nervous necrosis virus.

A real-time genotype-specific polymerase chain reaction (PCR) assay combined with high-resolution melting (HRM) analysis was developed to assess the most common genotypes of nervous necrosis viruses or nodaviruses. Nodaviruses are the causal agents of viral nervous necrosis infections, which have been wreaking havoc in the aquaculture industry worldwide, with fish mortality up to 100%. The four different genotypes of nodaviruses correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostics requires analysis of genetic variation among viruses. The aim of the present study was to develop a real-time tetra-primer genotype-specific PCR assay for genotype identification. Four primers were utilized for simultaneous amplification of nodavirus genotype-specific products in a single closed-tube PCR after a reverse-transcription reaction using RNA isolated from fish samples. For high-throughput sample analysis, SYBR Green-based real-time PCR was used in combination with HRM analysis. The assay was evaluated in terms of specificity and sensitivity. The analysis resulted in melting curves that were indicative of each genotype. The detection limit when using reference plasmids was 100 ag/µL for both genotypes, while the sensitivity of the assays when testing a complex mixture was 10 fg/µL for red-spotted grouper nervous necrosis virus (RGNNV) and 100 fg/µL for striped jack nervous necrosis virus (SJNNV). To test the capability of this method under real-world conditions, 58 samples were examined. All samples belonged to the RGNNV genotype, which was fully validated. The results were in full agreement with genotyping by reference methods. The proposed methodology provides a rapid, sensitive, specific, robust and automatable assay for nodavirus genotyping, making it a useful tool for diagnosis and screening for epidemiological studies.

ssRNA viruses from biotrophic Oomycetes form a new phylogenetic group between Nodaviridae and Tombusviridae.

Plasmopara halstedii virus (PhV) is one of the few characterized oomycete viruses. Although it is fully sequenced and well-studied in its genetic diversity, the exact classification and phylogenetic relationships of PhV remain uncertain. The only known virus with characteristics similar to PhV is the Sclerophthora macrospora Virus A (SmV-A). Both viruses infect obligate biotrophic oomycetes. While RNA-dependent RNA polymerases (RdRp) of oomycetes viruses have high similarity to the corresponding enzymes from viruses classified in the family Nodaviridae, the coat proteins (CP) seem to be completely different from those of other viruses of this family. In contrast, the coat proteins of PhV and SmV-A have high similarity to viruses classified in the Tombusviridae, Circoviridae and a new group of hybrid DNA-RNA viruses (so-called chimeric viruses or cruciviruses). Because phylogenetic analyses based on the sequences of either RdRp or CP result in different affinities, an alternative, genome-based approach combining the sequences of both proteins was used. This analysis placed the two oomycete viruses together with Tombunodavirus UC1 in a new, independent group between families Nodaviridae and Tombusviridae.

Viral encephalopathy and retinopathy in aquaculture: a review.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a major devastating threat for aquatic animals. Betanodaviruses have been isolated in at least 70 aquatic animal species in marine and in freshwater environments throughout the world, with the notable exception of South America. In this review, the main features of betanodavirus, including its diversity, its distribution and its transmission modes in fish, are firstly presented. Then, the existing diagnosis and detection methods, as well as the different control procedures of this disease, are reviewed. Finally, the potential of selective breeding, including both conventional and genomic selection, as an opportunity to obtain resistant commercial populations, is examined.

SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system.

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Paper-based archiving of biological samples from fish for detecting betanodavirus.

This study was carried out to evaluate the efficiency of the Flinders Technology Associates (FTA(®)) card (Whatman(®)) as a sampling device and storage platform for RNA from betanodavirus-infected biological samples (viz., larvae, broodstock, cell culture supernatants and rearing seawater spiked with infected materials). The study showed that FTA cards can be used to detect betanodaviruses by reverse transcription-polymerase chain reaction (RT-PCR). The diagnostic efficiency of RT-PCR from all sample types on FTA cards decreased after 21 days of storage at 4 °C, although the virus could be detected up to 28 days by nested RT-PCR. The FTA card protocol thus provides a supplementary method for quick and easy collection of samples, preservation of RNA on a dry storage basis, and detection of betanodavirus-infected fish.

Divergence and codon usage bias of Betanodavirus, a neurotropic pathogen in fish.

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.

Epidemiological characterization of VNNV in hatchery-reared and wild marine fish on Hainan Island, China, and experimental infection of golden pompano (Trachinotus ovatus) juveniles.

The current epidemiological situation of viral nervous necrosis virus (VNNV) on Hainan Island was investigated. A total of 490 hatchery-reared fish and 652 wild fish were sampled for VNNV detection from March 2013 to May 2014. Positive detection rates of 84.53% (153/181) and 0.97 % (3/309) were obtained in diseased and healthy hatchery-reared samples, respectively, by conventional RT-PCR. However, using more-sensitive nested RT-PCR, the positive detection rates in healthy hatchery-reared fish reached up to 64.08% (198/309), suggesting that asymptomatic VNNV carriers commonly exist among larvae and juveniles breeding on Hainan Island. In wild-fish samples, 2.6% (17/652) and 34.2% (223/652) positive detection rates were observed using RT-PCR and nested RT-PCR, respectively, indicating that wild fish may be a potential reservoir for VNNV. Phylogenetic analysis showed that all 52 VNNV isolates from cultured fish belong to the RGNNV genotype, but 2 out of 48 VNNV isolates from wild fish samples were found to be of the SJNNV genotype. This study is the first to confirm the existence of SJNNV-genotype VNNV in China. Golden pompano, an important fish species for culture, was selected as a fish model to investigate the optimal conditions for RGNNV disease progression in artificial infection experiments. The effects of temperature, salinity, and fish size were evaluated. Results showed that 28 °C and 20 ‰ are the optimal infection temperature and salinity, respectively, and golden pompano juveniles with small body sizes are more susceptible to RGNNV. These findings are highly consistent with those conditions involved in the natural outbreak of RGNNV.

Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.

Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm-raised tilapia Oreochromis niloticus (L.) in Thailand.

Betanodavirus infection was diagnosed in larvae of farm-raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous necrosis virus (RGNNV).

Infections of nervous necrosis virus in wild and cage-reared marine fish from South China Sea with unexpected wide host ranges.

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage-reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage-reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage-reared fish, respectively. However, by nested RT-PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage-reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage-reared fish by seminested PCR assay, respectively. However, by nested RT-PCR, the positive signal was observed in 65.2% and 100% species of wild and cage-reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty-five species of the marine fish were the new hosts of NNV.

A new nodavirus is associated with covert mortality disease of shrimp.

A new nodavirus, named covert mortality nodavirus (CMNV), is associated with covert mortality disease of shrimp which has caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by reverse transcription (RT)-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in the hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in the muscle and lymphoid organ. The tubular epithelium of the hepatopancreas showed significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shared 54, 53 and 39% identity with the amino acid sequences of RNA-dependent RNA polymerase from Flock House virus, black beetle virus and MrNV. The results of fluorescence in situ hybridization showed that the hepatopancreas, striated muscle and lymphoid organ were positively reacting tissues. The mean size of negative-stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV, and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV-positive.

Genomic classification of betanodavirus by molecular phylogenetic analysis of the coat protein gene.

The classification of betanodavirus into four species was reviewed including newer and well-characterised isolates. Six major clusters were identified, four of which were similar to the classic species. Two single isolate clusters were worth consideration as new species.