Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR

A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed

Antiviral immune response in fish and shellfish

Innate immunity constitutes the main defense mechanism in fish and shellfish, which is the most antique and efficient system to protect organisms against microbial infections. In addition, lower vertebrates have less evolved adaptive immune responses compared to higher vertebrates, and therefore, rely on innate immune defenses for the control of pathogens (1). Nonetheless, it is worth noting that the contribution of both innate and adaptive immune responses leads to global protection against pathogens in the organism. The objective of this Research Topic is to reflect the significant advances in the study of the immune response against viral infections in fish. This topic contributes to identifying new mechanisms of response to infections and new effector molecules against viral pathogens. In addition, these studies provide a further understanding of the resistance of lower vertebrates to pathogens infection as well as to the prevention of exacerbated immune responses. The contributing articles collected in this Research Topic are grouped into the following proposed sections related to the antiviral defense in fis

Expression and antiviral activity of a β-defensin-like peptide identified in the rainbow trout (Oncorhynchus mykiss) EST sequences

The in silico identification of a β-defensin-like peptide sequence (omBD-1) in the rainbow trout (Oncorhynchuss mykiss) database of salmonid EST is reported here. We have studied the transcript expression of this β-defensin-like sequence in different organs and expressed the recombinant peptide in a fish cell line. Finally, we have demonstrated the in vitro antiviral activity of the recombinant trout β-defensin-like peptide against viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most devastating viruses for worldwide aquaculture. Thus, the resistance to VHSV infection of EPC cells transfected with pMCV 1.4-omBD-1 has been shown. Since EPC cells transfected with omBD-1 produced acid and heat stable antiviral activity and up regulation of Mx, a type I IFN-mediated mechanism of antiviral action is suggested. To our knowledge, this is the first report showing biological activity of a β-defensin-like peptide from any fish

The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences

A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5′ upstream sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp β-actin gene (pAE6-GVHSV) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-GVHSV). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-GVHSV. However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation

Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms

Download PDFsettingsOrder Article Reprints Open AccessCommunication Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms by Maria Salvador-Mira 1,2,Veronica Chico 1,2ORCID,Monica Arostica 3,Fanny Guzmán 3ORCID,Nerea Roher 4,Luis Perez 1,2ORCID andMaria del Mar Ortega-Villaizan 1,2,*ORCID 1 Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández (IBMC-UMH), 03202 Elche, Spain 2 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández (IDiBE-UMH), 03202 Elche, Spain 3 Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile 4 Department Biologia Cellular, Fisiologia Animal i Immunologia, Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain * Author to whom correspondence should be addressed. Int. J. Mol. Sci. 2021, 22(21), 11821; https://doi.org/10.3390/ijms222111821 Submission received: 1 October 2021 / Revised: 24 October 2021 / Accepted: 26 October 2021 / Published: 30 October 2021 (This article belongs to the Section Biochemistry) Downloadkeyboard_arrow_down Browse Figures Versions Notes Abstract One of the challenges of science in disease prevention is optimizing drug and vaccine delivery. Until now, many strategies have been employed in this sector, but most are quite complex and labile. To overcome these limitations, great efforts are directed to coupling drugs to carriers, either of natural or synthetic origin. Among the most studied cell carriers are antigen-presenting cells (APCs), however, red blood cells (RBCs) are positioned as attractive carriers in drug delivery due to their abundance and availability in the body. Furthermore, fish RBCs have a nucleus and have been shown to have a strong involvement in modulating the immune response. In this study, we evaluated the binding of three peptides to rainbow trout RBCs, two lectin-like peptides and another derived from Plasmodium falciparum membrane protein, in order to take advantage of this peptide-RBCs binding to generate tools to improve the specificity, efficacy, immunostimulatory effect, and safety of the antiviral therapeutic or prophylactic administration systems currently used

IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells

Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, th

Development of new therapeutical/adjuvant molecules by pepscan mapping of autophagy and IFN inducing determinants of rhabdoviral G proteins

Surface glycoproteins of enveloped virus are potent elicitors of both innate and adaptive host immune responses. Therefore, the identification of viral glycoprotein determinants directly implicated in the induction of these responses might be of special interest for designing new therapeutical/adjuvant molecules. In this work we review the contribution of the “pepscan” approach to the screening of viral functions in the sequence of glycoprotein G (gpG) of the fish rhabdovirus of viral hemorrhagic septicemia (VHSV). Among others, by scanning gpG peptides, it has been possible to identify and validate minimal determinants for gpG directly implicated in initiating the fish type I Interferon-associated immune responses as well as in the antiviral autophagy program. Further fine-tunning of the identified peptides in the gpG of VHSV has allowed designing novel adjuvants that decrease DNA vaccine requirements and identify possible innovative antiviral molecules. In addition, these results have also contributed to improve our knowledge on how to stimulate the fish immune system

In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV)-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of GVHSV protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9–CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding GVHSV gene are due to GVHSV rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants