A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

Detection of SJNNV and RGNNV genotypes using a relative quantification RT‐PCR assay

Viral Encephalopathy and Retinopathy (VER), is caused by a nodavirus included within the Betanodavirus genus of the Nodaviridae family. This disease affects more than 30 marine fish species worldwide and has been a major obstacle in the aquaculture industry; control of the disease is based on virus detection, essentially in carrier specimens. This study describes a real time PCR procedure for viral nervous necrosis virus detection from several organs of sea bass, Senegalese sole, and gilt‐head sea bream, from fish displaying either clinical symptoms or asymptomatic cases. The sensitivity of this technique was about 10⁶‐fold higher than that of the conventional RT‐PCR. The newly designed primers detected nodavirus isolates belonging to the RGNNV and SJNNV genotypes.     

Comparisons among the complete genomes of four betanodavirus genotypes

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes and have been classified (based on analysis of RNA2 sequences) into 4 genotypes: tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV), striped jack nervous necrosis virus (SJNNV), and redspotted grouper nervous necrosis virus (RGNNV). Full-length genomes of TPNNV and BFNNV were sequenced for the first time in this study. Their sequence data and those of SJNNV and RGNNV retrieved from GenBank were compared in order to investigate the relationships among the 4 genotypes. Between TPNNV and BFNNV, sequence identities were relatively high in RNA1 and encoded Protein A, but were not significantly high in RNA2 or the coat protein (CP). Similarly, between BFNNV and RGNNV, the amino acid sequences of CP were highly similar, but identities of RNA1, RNA2, and Protein A sequences were not especially high. Furthermore, multiple alignment data of the 4 genotypes of RNA2 sequences revealed that the TPNNV and SJNNV sequences have the same sizes of gaps and extra sequences at the same positions. Collectively, these apparent contradictions in sequence identity suggest that betanodavirus genomes have been constructed via complex evolutionary processes.     

Metabolic profiles of fish nodavirus infection in vitro: RGNNV induced and exploited cellular fatty acid synthesis for virus infection

Red‐spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis disease, has caused high mortality and heavy economic losses in marine aquaculture worldwide. However, changes in host cell metabolism during RGNNV infection remain largely unknown. Here, the global metabolic profiling during RGNNV infection and the roles of cellular fatty acid synthesis in RGNNV infection were investigated. As the infection progressed, 71 intracellular metabolites were significantly altered in RGNNV‐infected cells compared with mock‐infected cells. The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased, whereas those that correlated with fatty acid synthesis were significantly up‐regulated during RGNNV infection. Among them, tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection. In addition, RGNNV infection induced the formation of lipid droplets and re‐localization of fatty acid synthase (FASN), indicating that RGNNV induced and required lipogenesis for viral infection. The exogenous addition of palmitic acid (PA) enhanced RGNNV infection, and the inhibition of FASN and acetyl‐CoA carboxylase (ACC) significantly decreased RGNNV replication. Additionally, not only inhibition of palmitoylation and phospholipid synthesis, but also destruction of fatty acid β‐oxidation significantly decreased viral replication. These data suggest that cellular fatty acid synthesis and mitochondrial β‐oxidation are essential for RGNNV to complete the viral life cycle. Thus, it has been demonstrated for the first time that RGNNV infection in vitro overtook host cell metabolism and, in that process, cellular fatty acid synthesis was an essential component for RGNNV replication.     

Serological relationships among genotypic variants of betanodavirus

Betanodaviruses, the causative agents of viral nervous necrosis or viral encephalopathy and retinopathy, are divided into 4 genotypes based on the coat protein gene (RNA2). In the present study, serological relationships among betanodavirus genotypic variants were examined by virus neutralization tests using rabbit antisera raised against purified virions of strains representative of each genotype. All 20 isolates examined shared epitopes for neutralizing, but they fell into 3 major serotypes (A, B, C). This sero-grouping is in part consistent with their genotypes, i.e. Serotype A for striped jack nervous necrosis virus (SJNNV) genotype, Serotype B for tiger puffer nervous necrosis virus (TPNNV) genotype, and Serotype C for both redspotted grouper nervous necrosis virus (RGNNV) and barfin flounder nervous necrosis virus (BFNNV) genotypes. The serological relatedness between RGNNV and BFNNV genotypes may result from their relatively higher similarity in RNA2 sequences. In neutralization tests using antisera of kelp grouper Epinephelus moara, which were raised against recombinant coat proteins representing each genotype, anti-SJNNV and anti-TPNNV sera neutralized only the homologous strain, and anti-RGNNV and anti-BFNNV sera reacted with both RGNNV and BFNNV strains. The present serological findings will be important in investigating the infectivity and host-specificity of betanodaviruses and in developing vaccines for the disease.     

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.     

Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV.

Immunisation by intraperitoneal injection of an oil-emulgated recombinant partial capsid protein (rT2) from striped jack nervous necrosis virus (SJNNV) was performed on adult turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus. A specific humoral immune response was recorded in both species, and the levels of rT2-specific antibodies increased markedly in all groups during the 20 wk experiment. A challenge model for SJNNV was established by intramuscular injection of juvenile turbot. The turbot developed viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN), with cumulative mortality in the range of 25 to 66%, after intramuscular inoculation with SJNNV propagated in the striped snake head cell line (SSN-1). Although neither clinical signs nor mortality were registered, SJNNV was neuroinvasive after bath exposure. The infection after both modes of challenge was verified by means of immunohistochemistry and RT-PCR, and SJNNV was reisolated in cell culture. The results indicate that SJNNV may have entered the central nervous system (CNS) by axonal transport through motor nerves after intramuscular inoculation. A vaccine efficacy test was performed on juvenile turbot, employing oil emulsified rT2 as a test vaccine and intramuscular inoculation of SJNNV. Significant protection was observed when the challenge was performed 10 wk post-vaccination.     

Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the ₁₉₅VNVSVLCR₂₀₂ motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine₁₉₇, Valine₁₉₉, and Cysteine₂₀₁ are critical for antibody binding. Substitution of Leucine₂₀₀ in the RGNNV, BFNNV, and TPNNV genotypes with Methionine₂₀₀ (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.     

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV)

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.     

Multiple infection dynamics has pronounced effects on the fitness of RNA viruses

Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two‐fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka‐Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed.     

The Role of Virulence in In Vivo Superinfection Fitness of the Vertebrate RNA Virus Infectious Hematopoietic Necrosis Virus

We have developed a novel in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a coevolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 h to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal-virulence and unequal-virulence genotype pairs, the frequency of superinfection in most cases was the same regardless of which genotype was used in the primary exposure. The ability to replicate in the context of superinfection also did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural vertebrate host.     

Asymmetric competitive suppression between strains of dengue virus

Background  

Within-host competition between strains of a vector-borne pathogen can affect strain frequencies in both the host and vector, thereby affecting viral population dynamics. However little is known about inter-strain competition in one of the most genetically diverse and epidemiologically important mosquito-borne RNA virus: dengue virus (DENV). To assess the strength and symmetry of intra-host competition among different strains of DENV, the effect of mixed infection of two DENV serotypes, DENV2 and DENV4, on the replication of each in cultured mosquito cells was tested. The number of infectious particles produced by each DENV strain in mixed infections was compared to that in single infections to determine whether replication of each strain was decreased in the presence of the other strain (i.e., competition). The two DENV strains were added to cells either simultaneously (coinfection) or with a 1 or 6-hour time lag between first and second serotype (superinfection).     

Patterns of accumulation of Bean common mosaic virus in Phaseolus vulgaris genotypes nearly isogenic for the I locus

The I locus of Phaseolus vulgaris is genetically and phenotypically well described, conferring incompletely dominant, temperature‐dependent resistance against viruses currently assigned to at least four Potyvirus species. Despite the fact that the resistance allele at this locus, the I gene, has been incorporated into nearly all bean germplasm worldwide, little is known regarding its resistance mechanism. In the present study, P. vulgaris lines nearly isogenic for I were challenged with Bean common mosaic virus (BCMV; genus Potyvirus) in order to investigate at the cellular level the temperature‐dependent resistance reaction. Immunolocalisation and confocal laser scanning microscopy were employed to visualise the virus and to identify patterns of BCMV accumulation in resistant, susceptible and heterozygous genotypes. Virus was detected in all three genotypes regardless of temperature, supporting previous findings that BCMV accumulates in protoplasts containing the I allele. Genotype‐specific and temperature‐specific patterns of virus accumulation suggested a resistance mechanism that depends on host recognition of viral replication and/or local movement.     

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Whether viral pathogens that induce ER stress responses benefit the host or the virus remains controversial. In this study we show that betanodavirus induced ER stress responses up-regulate GRP78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. Betanodavirus (redspotted grouper nervous necrosis virus, RGNNV) infection resulted in the following increased ER stress responses in fish GF-1 grouper fin cells: (1) IRE-1 and ATF-6 sensors at 48 h post-infection (p.i.) that up-regulated chaperone protein GRP78; (2) activation of caspase-12; and (3) PERK phosphorylation and down-regulation of Bcl-2. Analyses of GRP78 functions during viral replication using either loss-of-function or gain-of-function approaches showed that GRP78 over-expression also enhanced viral replication and induced cell death. Then, we found that zfGRP78 localization gradually increased in mitochondria after RGNNV infection by EGFP tagging approach. Furthermore, zfGRP78 can interact with viral RNA-dependent RNA polymerase (RdRp) by using immunofluorescent and immunoprecipitation assays. Finally, we found that blocking GRP78-mediated ER signals can reduce the viral death factors protein α and protein B2 expression and decrease the Bcl-2 down-regulation mediated mitochondria-dependent cell death, which also enhances host cellular viability. Taken together, our results suggest that RGNNV infection and expression can trigger ER stress responses, which up-regulate the chaperone GRP78 at early replication stage. Then, GRP78 can interact with RdRp that may enhance the viral replication for increasing viral death factors’ expressions at middle-late replication stage, which can enhance mitochondrial-mediated cell death pathway and viral spreading. These results may provide new insights into the mechanism of ER stress-mediated cell death in RNA viruses.     

Unique genome replication mechanism of the archaeal virus AFV1

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two‐dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D‐loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark‐field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.     

Cloning of the fish cell line SSN-1 for piscine nodaviruses

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and *BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degrees C for strain SGWak97 (RGNNV), 20 to 25 degrees C for strain SJNag93 (SJNNV), 20 degrees C for strain TPKag93 (TPNNV), and 15 to 20 degrees C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line.