Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication.

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp.

Lysyl-tRNA synthetase produces diadenosine tetraphosphate to curb STING-dependent inflammation.

Inflammation is an essential part of immunity against pathogens and tumors but can promote disease if not tightly regulated. Self and non-self-nucleic acids can trigger inflammation, through recognition by the cyclic GMP-AMP (cGAMP) synthetase (cGAS) and subsequent activation of the stimulator of interferon genes (STING) protein. Here, we show that RNA:DNA hybrids can be detected by cGAS and that the Lysyl-tRNA synthetase (LysRS) inhibits STING activation through two complementary mechanisms. First, LysRS interacts with RNA:DNA hybrids, delaying recognition by cGAS and impeding cGAMP production. Second, RNA:DNA hybrids stimulate LysRS-dependent production of diadenosine tetraphosphate (Ap4A) that in turn attenuates STING-dependent signaling. We propose a model whereby these mechanisms cooperate to buffer STING activation. Consequently, modulation of the LysRS-Ap4A axis in vitro or in vivo interferes with inflammatory responses. Thus, altogether, we establish LysRS and Ap4A as pharmacological targets to control STING signaling and treat inflammatory diseases.

Vaccination of carp against SVCV with an oral DNA vaccine or an insect cells-based subunit vaccine.

We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV.

Zebrafish Plzf transcription factors enhance early type I IFN response induced by two non-enveloped RNA viruses.

The BTB-POZ transcription factor Promyelocytic Leukemia Zinc Finger (PLZF, or ZBTB16) has been recently identified as a major factor regulating the induction of a subset of Interferon stimulated genes in human and mouse. We show that the two co-orthologues of PLZF found in zebrafish show distinct expression patterns, especially in larvae. Although zbtb16a/plzfa and zbtb16b/plzfb are not modulated by IFN produced during viral infection, their over-expression increases the level of the early type I IFN response, at a critical phase in the race between the virus and the host response. The effect of Plzfb on IFN induction was also detectable after cell infection by different non-enveloped RNA viruses, but not after infection by the rhabdovirus SVCV. Our findings indicate that plzf implication in the regulation of type I IFN responses is conserved across vertebrates, but at multiple levels of the pathway and through different mechanisms.

Zebrafish ISG15 exerts a strong antiviral activity against RNA and DNA viruses and regulates the interferon response.

ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.

T cell diversity and TcR repertoires in teleost fish.

In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses.

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.

Viral haemorrhagic septicaemia virus induces vig-2, a new interferon-responsive gene in rainbow trout.

An mRNA differential display methodology was used to study the rainbow trout response to viral infection. A new transcript (vig-2) induced by viral haemorrhagic septicaemia virus (VHSV) in rainbow trout leucocytes was identified from the head-kidney. vig-2 was also induced in vivo during experimental infection and following DNA immunisation with a plasmid containing a gene encoding the viral glycoprotein. Viral induction of vig-2 was blocked by cycloheximide (CHX), indicating its dependency on a newly synthesised intermediate protein. This intermediate protein is most probably related to interferon because treatment of cells with a conditioned medium displaying an interferon-like activity resulted in a strong vig-2 expression, which was not blocked by CHX treatment. The cDNA sequence of the vig-2 transcript displays several mRNA destabilisation motifs and two signals characteristic of immediate-early gene expression. Curiously, vig-2 has no evident encoding potential except for a small 51 amino acid putative polypeptide with no clear similarity to any sequence available in the databanks. Therefore, the complete vig-2 genomic sequence was determined from a lambda phage clone retrieved from a genomic DNA library of rainbow trout. The genomic organisation of vig-2 shows five exons delimited with typical splice acceptor and donor sites. A promoter with a canonical ISRE, confirming that vig-2 is an interferon-responsive gene, is also present 115 nt upstream of the first exon.

vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family.

We used mRNA differential display methodology to analyze the shift of transcription profile induced by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in rainbow trout leukocytes. We identified and characterized a new gene which is directly induced by VHSV. This VHSV-induced gene (vig-1) encodes a 348-amino-acid protein. vig-1 is highly expressed during the experimental disease in lymphoid organs of the infected fish. Intramuscular injection of a plasmid vector expressing the viral glycoprotein results in vig-1 expression, showing that the external virus protein is sufficient for the induction. vig-1 expression is also obtained by a rainbow trout interferon-like factor, indicating that vig-1 can be induced through different pathways. Moreover, vig-1 is homologous to a recently described human cytomegalovirus-induced gene. Accordingly, vig-1 activation may represent a new virus-induced activation pathway highly conserved in vertebrates. The deduced amino acid sequence of vig-1 is significantly related to sequences required for the biosynthesis of metal cofactors. This suggests that the function of vig-1 may be involved in the nonspecific virus-induced synthesis of enzymatic cofactors of the nitric oxide pathway.

Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout.

Glycoprotein (G) of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) contains several neutralizing epitopes. However, recombinant G protein never matches intact viral particles for immunogenicity. DNA immunization offers the possibility to deliver the antigen through the cellular machinery, thus mimicking natural infection. We constructed pCDNA gVHS and pCDNA gIHN plasmids with the G gene of VHSV and IHNV under the control of the CMV promoter, and we tested the plasmids for the accurate G protein expression prior to their use in fish immunization. Following intramuscular injection to adult rainbow trout, plasmid DNA was found inside the muscle cells shortly after injection and was still present 45 days later. mRNA of the G protein was detected in muscle tissue extracts, and the G protein was found within muscle cells at the site of injection. This resulted in the synthesis of high levels of specific neutralizing and protective antibodies. Fish injected with pCDNA gVHS and pCDNA gIHN in combination responded similarly to fish receiving one recombinant plasmid. In addition to the elicitation of a strong humoral response, DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms, since mRNAs of MHC class II and Mx were strongly activated at the site of injection.

The lambda B cell repertoire of kappa-deficient mice.

Analysis of the B cell repertoire is complicated by the huge diversity inherent in the germ line determined combinatory. Making use of knockout technology, kappa-deficient mice have been obtained. They constitute a shrewd model to follow the expression of an Ig minilocus, such as the lambda one, in the normal condition compared with classical transgenic models. Indeed, in contrast to wild type mice, in which only 5% of lambda B cells are produced, these mutant mice exclusively produce lambda positive B cells. Although, the lambda locus is well characterized and has a relatively simple organization, the mechanistic and selective pressures that govern its utilization are still poorly understood. The analysis of the lambda B cell repertoire in kappa-deficient mice, should therefore bring more conclusive informations. Here we present the lambda subtype distribution in the various cellular compartments of the kappa-deficient mice, and discuss the rules that can be responsible for this distribution. Our recent data indicate that the lambda subtype proportions in the bone marrow and the spleen result, for the major part, from mechanistic processes (i.e., recombinase accessibility, production of V-J functional joint and H/L pairings) while the lambda proportions found in the peritoneal cavity ensue from selective processes. Finally, the capacity to respond to various antigens is discussed from such a generated lambda B cell repertoire.

Various V-J rearrangement efficiencies shape the mouse lambda B cell repertoire.

The diversity of the B cell repertoire of C kappa knockout mice is limited by the expression of four lambda light chain types. Among the spleen B cells, lambda 1 is expressed by the majority (58%) of cells, and lambda 3 by the minority (8%), while lambda 2 (V2) and lambda 2 (Vx) are expressed in intermediate quantities (18% and 16%, respectively). To assess the influence of mechanistic pressures on the lambda subtype distribution, the proportions of the different lambda rearrangements were determined in various B cell subpopulations divided on the basis of the lambda subtype expressed, and the V lambda J lambda junction sequences were studied at different steps of B cell differentiation (pre-B, immature and mature B cells). The data show that (1) the ratio of productive/non-productive VJ junctions is determined by the nature of the lambda segments that are rearranged as can be observed in the pre-B cells, (2) V1-J1 non-productive rearrangements are often found in the lambda 1-negative B cells in the periphery, and (3) V1J3 junctions are often non-productive regardless of the nature of the cells analyzed. Our results, therefore, suggest that a strong probability of initiating a V1-J1 rearrangement and a weak probability of giving a productive V1J3 junction are responsible for the lambda 1 dominance and the lambda 3 under-expression, respectively. The intermediate proportion of lambda 2(V2) subtype is most likely due to a probability of obtaining a productive joint that is better than that for V1J3 and a probability of initiating a rearrangement that is lower than that for V1J1. However, the lambda 2(Vx) cell proportion cannot be determined only by these parameters.

Conserved distribution of lambda subtypes from rearranged gene segments to immunoglobulin synthesis in the mouse B cell repertoire.

The immunoglobulin lambda light chain system displays a limited diversity in inbred mice. Indeed, the lambda locus is organized in two recombination units: V lambda 2-V lambda x-J lambda 2-C lambda 2-psi J lambda 4-psi C lambda 4, which can produce either lambda 2(V2) or lambda 2(Vx) chains; and V lambda 1-J lambda 3-C lambda 3-J lambda 1-C lambda 1, which can produce either lambda 1 or lambda 3 chains. Each of these units is associated with an enhancer, E lambda 2-4 or E lambda 1-3, at the 3' side. The expression of each lambda chain is, therefore, controlled by distinct promoter and/or enhancer regions. To clarify the basis of these controls, we measured, by quantitative polymerase chain reaction, the proportions of each lambda subtype in BALB/c spleen mRNA and among genomic rearrangements. It appears that these distributions are similar to and consistent with the relative cellular frequencies in the spleen, as evaluated by flow cytometry. These results suggest that, in resting cells, the transcription rates are identical, regardless of the lambda subtype. After lipopolysaccharide (LPS) stimulation, the transcription rates per cell remain similar for all lambda subtypes despite different regulatory sequences. To detect eventual post-transcriptional regulations, we estimated the lambda light chain distribution in IgM secreted by LPS-stimulated B cells and in serum IgG. These distributions are still similar to those of lambda-expressing cells, lambda mRNA or genomic rearrangements. We conclude that the lambda subtype distribution is conserved from productive V-J rearranged genes to secreted lambda immunoglobulins, despite different regulatory sequences.

Mechanistic and selective constraints act on the establishment of V lambda J lambda junctions in the B cell repertoire.

Only four different subtypes of lambda Ig chains have been described in the mouse: lambda 1, lambda 2(V2), lambda 2(Vx), and lambda 3. These chains are encoded by gene segments all sequenced and well localized in chromosome 16. Although the lambda Ig system is both simple and well characterized, no exhaustive analysis has been done concerning V lambda J lambda junctions in nonintentionally stimulated B cells. To get an insight into the lambda B cell repertoire, we analyzed a large number of V lambda J lambda rearrangements isolated from spleen mRNA or genomic DNA of unimmunized adult BALB/c mice. By PCR amplification, more than 160 clones were obtained covering all V lambda J lambda recombinations. Simple recombinations of trimmed gene segments explain most sequences. Certain junctions have been found to be prevalent in each subtype, and an analysis of V lambda J lambda recombination sites shows that the splenic lambda repertoire can result from both differential efficiencies of rearrangement and selective processes.