Broadly cross-reactive immune responses in chickens immunized with chimeric virus-like particles of nodavirus displaying the M2e originated from avian and human influenza A viruses.

Avian influenza A viruses (IAVs) pose a persistent threat to poultry industry worldwide, despite the presence of vaccines. Additionally, reverse-zoonosis transmission potentially introduces human-originated IAVs into poultry and complicates the efforts to control the spread of influenza. Current avian influenza vaccines are primarily based upon the rapidly mutating hemagglutinin (HA) and neuraminidase (NA) glycoproteins, which limit their efficacy against diverse strains of IAVs. Hence, the highly conserved ectodomains of matrix 2 protein (M2e) of IAVs are widely studied as alternatives to the HA and NA. However, the differences in the M2e amino acid sequences between avian and human IAVs generate antibodies that do not cross-react reciprocally with IAVs from other origins. To broaden and enhance the immunogenicity of M2e, we fused two copies each of the M2e derived from avian and human IAVs at the C-terminal end of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (NvC). Transmission electron microscopic and dynamic light scattering analyses revealed that the chimeric protein self-assembled into virus-like particles (VLPs). Immunization of chickens with the chimeric VLPs demonstrated a robust induction of broadly reactive immune responses against both the M2e of avian and human IAVs. Additionally, the chimeric VLPs elicited the production of cytotoxic T lymphocytes (CTL), macrophages, as well as a well-balanced Th1 and Th2 population, indicating their potential in activating cell-mediated immune responses in chickens. Furthermore, the chimeric VLPs triggered the production of both Th1- and Th2-cytokines, attesting their potential in mounting a robust and balanced immune response in avian species. This study demonstrated the potential of these chimeric VLPs in stimulating and broadening cross-reactive immune responses in chickens against both avian and human IAVs.

1mΨ influences the performance of various positive-stranded RNA virus-based replicons.

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

The susceptibility of shi drum juveniles to betanodavirus increases with rearing densities in a process mediated by neuroactive ligand-receptor interaction.

Nervous necrosis virus (NNV) is one of the greatest threats to Mediterranean aquaculture, infecting more than 170 fish species and causing mortalities up to 100% in larvae and juveniles of susceptible species. Intensive aquaculture implies stressed conditions that affect the welfare of fish and their ability to fight against infections. In fact, a higher susceptibility to NNV has been related to poor welfare conditions. In order to analyze the physiological link between stressed conditions and increased susceptibility to NNV, as well as its possible role in the pathogenesis of this disease, we reared shi drum (Umbrina cirrosa) juveniles (30.7 ± 3.10 g body weight), which are expected to be asymptomatic upon NNV infection, at three stocking densities (2, 15, and 30 kg/m3) for 27 days and subsequently challenged them with NNV. We firstly characterized the stressed conditions of the specimens before and after infection and recorded the mortalities, demonstrating that stressed specimens reared at 30 kg/m3 suffered mortalities. However, the viral loads in different tissues were similar in all experimental groups, allowing horizontal and vertical transmission of the virus from asymptomatic specimens. All of these data suggest that shi drum tolerates wide ranges of culture densities, although high densities might be a setback for controlling NNV outbreaks in this species. In an attempt to understand the molecular pathways orchestrating this susceptibility change in stressed conditions, we performed a transcriptomic analysis of four tissues under mock- and NNV-infected conditions. In addition to the modification of the exceptive pathways such as cell adhesion, leukocyte migration, cytokine interaction, cell proliferation and survival, and autophagy, we also observed a heavy alteration of the neuroactive ligand-receptor pathway in three of the four tissues analyzed. Our data also point to some of the receptors of this pathway as potential candidates for future pharmacological treatment to avoid the exacerbated immune response that could trigger fish mortalities upon NNV infection.

Identification and functional characterization of fish IL-17 receptors suggest important roles in the response to nodavirus infection.

Th17 is a lymphocyte T helper (Th) subpopulation relevant in the control and regulation of the immune response characterized by the production of interleukin (IL)-17. This crucial cytokine family acts through their binding to the IL-17 receptors (IL-17R), having up to six members. Although the biology of fish Th17 is well-recognized, the molecular and functional characterization of IL-17 and IL-17R has been limited. Thus, our aim was to identify and characterize the IL-17R repertory and regulation in the two main Mediterranean cultured fish species, the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Our in silico results showed the clear identification of six members in each fish species, from IL-17RA to IL-17RE-like, with well-conserved gene structure and protein domains with their human orthologues. All of them showed wide and constitutive transcription in naïve tissues but with highest levels in mucosal tissues, namely skin, gill or intestine. In leucocytes, T mitogens showed the strongest up-regulation in most of the il17 receptors though il17ra resulted in inhibition by most stimulants. Interestingly, in vivo nodavirus infection resulted in alterations on the transcription of il17 receptors. While nodavirus infection led to some increments in the il17ra, il17rb, il17rc and il17rd transcripts in the susceptible European sea bass, many down-regulations were observed in the resistant gilthead seabream. Our data identify the presence and conservation of six coding IL-17R in gilthead seabream and European sea bass as well as their differential regulation in vitro and upon nodavirus infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00225-1.

The RNA interference response to alphanodavirus replication in Phlebotomus papatasi sand fly cells.

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi-derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo-siRNA) pathway, with minimal engagement from the PIWI-interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus-derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo-siRNA pathway.

T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchus labrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

Positive-strand RNA virus genome replication organelles: structure, assembly, control.

Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.

Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

OBJECTIVE: We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. METHODS: An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. RESULT: The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. CONCLUSION: These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

Selection and characterization of ssDNA aptamer targeting Macrobrachium rosenbergii nodavirus capsid protein: A potential capture agent in gold-nanoparticle-based aptasensor for viral protein detection.

The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Cytotoxic activity and gene expression during in vitro adaptive cell-mediated cytotoxicity of head-kidney cells from betanodavirus-infected European sea bass.

Cell-mediated cytotoxicity (CMC) is essential in eradicating virus-infected cells, involving CD8+ T lymphocytes (CTLs) and natural killer (NK) cells, through the activation of different pathways. This immune response is well-studied in mammals but scarcely in teleost fish. Our aim was to investigate the adaptive CMC using head-kidney (HK) cells from European sea bass infected at different times with nodavirus (NNV), as effector cells, and the European sea bass brain cell line (DLB-1) infected with different NNV genotypes, as target cells. Results showed low and unaltered innate cytotoxic activity through the infection time. However, adaptive CMC against RGNNV and SJNNV/RGNNV-infected target cells increased from 7 to 30 days post-infection, peaking at 15 days, demonstrating the specificity of the cytotoxic activity and suggesting the involvement of CTLs. At transcriptomic level, we observed up-regulation of genes related to T cell activation, perforin/granzyme and Fas/FasL effector pathways as well as apoptotic cell death. Further studies are necessary to understand the adaptive role of European sea bass CTLs in the elimination of NNV-infected cells.

Nervous Necrosis Virus Modulation of European Sea Bass (Dicentrarchus labrax, L.) Immune Genes and Transcriptome towards Establishment of Virus Carrier State.

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

Rapid and effective detection of Macrobrachium rosenbergii nodavirus using a combination of nucleic acid sequence-based amplification test and immunochromatographic strip.

Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 â„ƒ for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 104 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.

Virus-like Particles of Nodavirus Displaying the Receptor Binding Domain of SARS-CoV-2 Spike Protein: A Potential VLP-Based COVID-19 Vaccine.

Since the outbreak of the coronavirus disease 2019 (COVID-19), various vaccines have been developed for emergency use. The efficacy of the initial vaccines based on the ancestral strain of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has become a point of contention due to the emergence of new variants of concern (VOCs). Therefore, continuous innovation of new vaccines is required to target upcoming VOCs. The receptor binding domain (RBD) of the virus spike (S) glycoprotein has been extensively used in vaccine development due to its role in host cell attachment and penetration. In this study, the RBDs of the Beta (ß) and Delta (δ) variants were fused to the truncated Macrobrachium rosenbergii nodavirus capsid protein without the protruding domain (CΔ116-MrNV-CP). Immunization of BALB/c mice with the virus-like particles (VLPs) self-assembled from the recombinant CP showed that, with AddaVax as an adjuvant, a significantly high level of humoral response was elicited. Specifically, mice injected with equimolar of adjuvanted CΔ116-MrNV-CP fused with the RBD of the ß- and δ-variants increased T helper (Th) cell production with a CD8+/CD4+ ratio of 0.42. This formulation also induced proliferation of macrophages and lymphocytes. Overall, this study demonstrated that the nodavirus truncated CP fused with the SARS-CoV-2 RBD has potential to be developed as a VLP-based COVID-19 vaccine.

Structural and functional characterization of RNA dependent RNA polymerase of Macrobrachium rosenbergii nodavirus (MnRdRp).

Macrobrachium rosenbergii is a highly valued farmed freshwater species and its production has been affected globally by white tail disease caused by M. rosenbergii nodavirus (MrNV). MrNV is a single stranded positive sense RNA virus encoding RNA-dependent RNA polymerase (RdRp) for genome replication. Due to its essentiality for pathogenesis, it is an important drug target. The domain prediction of the complete sequence revealed the presence of two enzymatic regions namely methyl transferase and RdRp separated by transmembrane region. The predicted three-dimensional (3D) structure of MnRdRp using AlphaFold 2 shows that the structure is composed of three major sub-domains common for other polymerases namely fingers, palm and thumb. Structural similarity search revealed its similarity with other flaviviridea members especially with BVDV RdRp (BvdvRdRp). The structure of fingers and palm sub-domains is more conserved than the thumb sub-domain. A small α-helix named 'priming helix' having conserve Tyr was identified at position 829-833 with a potential role in de novo initiation. Analysis of electrostatic potential revealed that nucleotide and template channels are electropositive. Metal binding residues were identified as Asp599, Asp704 and Asp705. The α and ß phosphates of incoming nucleotide interact with two Mn2+, Arg455 and Arg537. For recognition of 2'-OH of incoming rNTP, Asp604, Ser661 and Asn670 were identified which can form H-bond network with 2'-OH group. Docking study revealed that Dasabuvir can potentially inhibit MnRdRp. The study concluded that the overall structure and function of MnRdRp are similar to Flaviviridae polymerases and their inhibitors can work against this enzyme.Communicated by Ramaswamy H. Sarma.

RNA-Seq analysis of juvenile gilthead sea bream (Sparus aurata) provides some clues regarding their resistance to the nodavirus RGNNV genotype.

Gilthead sea bream (Sparus aurata) is considered an asymptomatic carrier for the nodavirus genotype affecting European sea bass (Dicentrarchus labrax), RGNNV. Only larvae and juveniles of sea bream have been found to be susceptible to the RGNNV/SJNNV reassortant. Nevertheless, the molecular bases of the high resistance of sea bream against RGNNV are not known, and the overall transcriptome response to the virus remains unexplored. In this work, we conducted the first RNA-Seq analysis of sea bream infected with RGNNV to elucidate the immune mechanisms involved in their resistance. Since we recently published the transcriptome response of sea bass infected with RGNNV, we wanted to take the same tissues (brain and head kidney) at the same time points (24 and 72 h postinfection) to conduct comparative analyses. Sea bream responded to RGNNV challenge with a powerful immune arsenal characterized by the high expression of a multitude of type I interferon-related genes, immune receptors and antigen presentation-related genes in both tissues. Moreover, complement-, coagulation- and angiogenesis-related genes were highly enriched in the head kidney at the earlier sampling point. Interestingly, despite the strong immune response found in the brain, inflammation seems to have been restrained, resulting in a neuroprotective scenario. While the response in sea bass was characterized by the activation of the stress axis, which could lead to immunosuppression and neuronal damage, genes involved in these processes were not modulated in sea bream. An efficient antiviral response accompanied by low inflammation and the absence of stimulation of the stress response seem to play a role in the success of sea bream in resisting RGNNV infection.

Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching.

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

Molecular characterization of the cytotoxic and regulatory T cell coreceptor (CRTAM), and its ligand CADM1, in the European seabass and gilthead seabream.

T cell activation is a multifaceted process that depends on the activation of the T cell receptor (TCR). However, other coreceptors are also strictly necessary to provide co-signals and modulate the immune response. However, to date, most of these coreceptors are unknown in fish or their information is very limited. Therefore, in this work, we have identified the cytotoxic and regulatory T cell molecule, CRTAM, and its ligand, the cell adhesion molecule 1, CADM1, in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata); and evaluated their transcriptional levels. Both putative proteins showed the canonical architecture observed in mammals, where CRTAM exhibited two immunoglobulin domains and CADM1, both the a and b forms, exhibited three of these domains. In addition, phylogeny and synteny analyses showed their conservation throughout vertebrate evolution. We found constitutive expression of all three genes, with crtam and cadm1a being predominant in immune tissues such as spleen, thymus and head-kidney (HK), while cadm1b expression was more limited to the brain. In vitro, only the T cell mitogen phytohemagglutinin (PHA) up-regulated the transcription of crtam and cadm1a in HK leucocytes. Nodavirus (NNV) infection elicited an up-regulation of crtam and cadm1a in brain and HK, appearing earlier in seabream than in seabass, which could explain the resistance of seabream to the development of nodavirus disease. In addition, they are up-regulated during the innate cell-mediated cytotoxic response in seabream but not in seabass. Altogether, our data seem to indicate that CRTAM is more related to the innate cytotoxicity in seabream and more in the specific and T cell-mediated cytotoxicity in seabass. Our results highlight the importance of CRTAM and CADM1 as important molecules in the activation of T lymphocytes in seabass and seabream, but further studies are needed.

Immunity elicited by AMP-encoding plasmids fails to increase the protection of European sea bass against nodavirus.

Antimicrobial peptides (AMPs) are a potent arm of the innate immune system that can directly kill pathogens and induce immunomodulation. In the marine aquaculture, European sea bass (Dicentrarchus labrax L.) is one of the most prosperous species but is highly susceptible to nodavirus (NNV), which produces high rates of mortality in larvae and juvenile stages. Thus, we aimed to evaluate whether AMPs exert immunomodulatory and/or NNV-preventive actions in sea bass. To do this, plasmids encoding the sea bass AMPs dicentracin (pDIC), beta-defensin (pDB1), hepcidin (pHAMP2) or NK-lysin (pNKL) were generated and intramuscularly injected into sea bass juveniles to evaluate their immunomodulatory and anti-NNV roles. Sea bass muscle transcribes the AMPs and produces an increase in their circulating levels, along with an increase of the antibacterial activity. Immune-related gene analysis revealed a great activation of the inflammatory response and the recruitment of neutrophilic granulocytes at the site of injection. However, AMP-encoding plasmids, namely pHAMP2, negatively affected to NNV disease by increasing fish mortality. In conclusion, plasmids encoding AMPs show immunostimulatory effects on European sea bass but do not improve the resistance to NNV.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model.

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6-24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.