Julio Coll

A comparative review on European-farmed finfish RNA viruses and their vaccines

The diseases causing the highest ecological and socio-economical impacts in European farmed finfish are produced by RNA viruses. Salmon, trout, sea bream, sea bass, carp and turbot, suffer viral nervous necrosis produced by betanodaviruses (VNNV), infectious pancreatic necrosis produced by aquabirnaviruses (IPNV), viral haemorrhagic septicemia (VHSV) and infectious haematopoietic necrosis (IHNV) produced by novirhabdoviruses, spring viremia of carp produced by vesicular-like rhabdoviruses (SVCV), salmon pancreas disease and trout sleeping disease produced by alphaviruses (SAV) and infectious salmon anaemia produced by isaviruses (ISAV). There are not yet any effective treatments other than destroying all fish in infected farms, avoiding fish movements to and from infected areas and, in some particular cases, vaccination. The comparative study of the molecular characteristics of those RNA viruses and the state of knowledge of their vaccines, point to the development of new DNA vaccines for some RNA viruses, design of new mass delivery methods, maternal transfer of immunity, more extensive crossprotection studies between genotypes, use of safer all-fish plasmid control elements and study of DNA plasmid distribution after vaccination, as some of the major gaps that need urgent filling. In addition, to obtain similar protection levels to those produced by viral infections in survivors, live attenuated and/or some oil-adjuvanted inactivated virus vaccines, molecular adjuvants and/or other viral components (dsRNA or viral proteins interfering with fish defences), might have to be included in new DNA vaccine formulations. Furthermore, to be approved by the corresponding European authorities, fish viral DNA vaccines would also require the study of the persistence in fish of the introduced DNA, their possible impact to the aquatic environment and the acceptance of potential consumers.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach

BackgroundDespite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV).ResultsTwo-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36).ConclusionsThe genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

DNA vaccination by immersion and ultrasound to trout viral haemorrhagic septicaemia virus

This work reports preliminary data on the application of a novel method, ultrasound, for the DNA vaccination of rainbow trout. First, the best formulations were selected that increased the transfer by immersion of a plasmid coding for the green fluorescent protein (GFP) gene into trout fry. Quantification of GFP expression by fluorescence in the fin cells was used to study time course, DNA concentration dependence and comparison of different formulations. The best GFP expression results were obtained with short pulses of ultrasound, DOTAP liposomes and recombinant bacteria or bactofection. Other liposomes or microencapsulation formulations resulted in a GFP fluorescence similar to background values. Second, DNA immersion-vaccination of immunocompetent fingerling trout with the selected formulations was performed by using a plasmid coding for the glycoprotein G gene of the viral haemorrhagic septicaemia virus (VHSV). The immunization of fingerling trout was estimated by measuring humoral antibody, lymphoproliferation and VHSV challenge responses. Short pulses of low intensity ultrasound were the only method by which both humoral antibody responses and survival after VHSV challenge were obtained. Immersion DNA-vaccination using short pulses of ultrasound could eventually lead to a practical way to vaccinate small fish.

Interleukin 8 and CK-6 chemokines specifically attract rainbow trout (Oncorhynchus mykiss) RTS11 monocyte–macrophage cells and have variable effects on their immune functions

In the current work, we have demonstrated that both rainbow trout (Oncorhynchus mykiss) interleukin 8 (IL-8), a CXC chemokine, and CK-6, a CC chemokine, are able of efficiently attracting RTS11, a rainbow trout established macrophage-monocyte-like cell line. Interestingly, two sub-populations of non-adherent cells are distinguishable by flow cytometry that could be identified as immature monocyte- and mature macrophage-like populations, respectively, and the two chemokines studied exert their effects on different populations. Although IL-8 specifically attracts the monocyte-like sub-population, CK-6 specifically attracts the macrophage-like cell sub-population. We have also determined the effects of both of these chemokines on RTS11 phagocytosis, respiratory burst and the expression of other immune-related genes. We found that IL-8 inhibited the phagocytosis capacity of RTS11 cells belonging to the macrophage-like profile. No effect was observed, however, on the respiratory burst, immune function that has been considerably affected throughout the establishment of the cell culture. Concerning the effect that IL-8 and CK-6 have on the expression of other immune genes, we found that IL-8 significantly induced the levels of expression of CK-6, IL-8, pro-inflammatory cytokines such as IL-1beta and tumour necrosis factor alpha (TNF-alpha) of RTS11 cells. On the other hand, CK-6 induced the levels of expression of IL-8, iNOS and the integrin CD-18, while it had very faint effect on pro-inflammatory cytokines. These results constitute one of the very few studies in which the effect of IL-8, a CXC chemokine, on monocyte-like cells is described. Moreover, it demonstrates that different monocyte-macrophage sub-populations have different reactivity to different chemokines.

Oral immunization of rainbow trout to infectious pancreatic necrosis virus (Ipnv) induces different immune gene expression profiles in head kidney and pyloric ceca

Induction of neutralizing antibodies and protection by oral vaccination with DNA-alginates of rainbow trout Oncorhynchus mykiss against infectious pancreatic necrosis virus (IPNV) was recently reported. Because orally induced immune response transcript gene profiles had not been described yet neither in fish, nor after IPNV vaccination, we studied them in head kidney (an immune response internal organ) and a vaccine entry tissue (pyloric ceca). By using an oligo microarray enriched in immune-related genes validated by RTqPCR, the number of increased transcripts in head kidney was higher than in pyloric ceca while the number of decreased transcripts was higher in pyloric ceca than in head kidney. Confirming previous reports on intramuscular DNA vaccination or viral infection, mx genes increased their transcription in head kidney. Other transcript responses such as those corresponding to interferons, their receptors and induced proteins (n=91 genes), VHSV-induced genes (n=25), macrophage-related genes (n=125), complement component genes (n=176), toll-like receptors (n=31), tumor necrosis factors (n=32), chemokines and their receptors (n=121), interleukines and their receptors (n=119), antimicrobial peptides (n=59), and cluster differentiation antigens (n=58) showed a contrasting and often complementary behavior when head kidney and pyloric ceca were compared. For instance, classical complement component transcripts increased in head kidney while only alternative pathway transcripts increased in pyloric ceca, different β-defensins increased in head kidney but remained constant in pyloric ceca. The identification of new gene markers on head kidney/pyloric ceca could be used to follow up and/or to improve immunity during fish oral vaccination.

Specific Regulation of the Chemokine Response to Viral Hemorrhagic Septicemia Virus at the Entry Site

ABSTRACT The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

New tools to study RNA interference to fish viruses : Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids, transfected into EPC cells and transformed clones were obtained by puromycin selection. Sequence-specific interference with VHSV could only be observed with EPC clones transformed with a mixture of the three shDNAs, rather than with those clones obtained with individual sh DNAs. However, interference was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.

Trout oral VP2 DNA vaccination mimics transcriptional responses occurring after infection with infectious pancreatic necrosis virus (IPNV)

Time-course and organ transcriptional response profiles in rainbow trout Oncorhynchus mykiss were studied after oral DNA-vaccination with the VP2 gene of the infectious pancreatic necrosis virus (IPNV) encapsulated in alginates. The profiles were also compared with those obtained after infection with IPNV. A group of immune-related genes (stat1, ifn1, ifng, mx1, mx3, il8, il10, il11, il12b, tnf2, mhc1uda, igm and igt) previously selected from microarray analysis of successful oral vaccination of rainbow trout, were used for the RTqPCR analysis. The results showed that oral VP2-vaccination qualitatively mimicked both the time-course and organ (head kidney, spleen, intestine, pyloric ceca, and thymus) transcriptional profiles obtained after IPNV-infection. Highest transcriptional differential expression levels after oral vaccination were obtained in thymus, suggesting those might be important for subsequent protection against IPNV challenges. However, transcriptional differential expression levels of most of the genes mentioned above were lower in VP2-vaccinated than in IPNV-infected trout, except for ifn1 which were similar. Together all the results suggest that the oral-alginate VP2-vaccination procedure immunizes trout against IPNV in a similar way as IPNV-infection does while there is still room for additional improvements in the oral vaccination procedure. Some of the genes described here could be used as markers to further optimize the oral immunization method.

Pepscan Mapping of Viral Hemorrhagic Septicemia Virus Glycoprotein G Major Lineal Determinants Implicated in Triggering Host Cell Antiviral Responses Mediated by Type I Interferon

ABSTRACT Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

Salmonid viral haemorrhagic septicaemia virus: fusion-related enhancement of virus infectivity by peptides derived from viral glycoprotein G or a combinatorial library.

To search for enhancers and/or inhibitors of viral haemorrhagic septicaemia virus (VHSV, a salmonid rhabdovirus) infectivity, a total of 51 peptides from a pepscan of viral envelope protein G, a recombinant peptide from protein G (frg11) and 80 peptide mixtures from an alpha-helix-favoured combinatorial library were screened. However, contrary to what occurs in many other enveloped viruses, only peptides enhancing rather than inhibiting VHSV infectivity were found. Because some of the enhancer pepscan G peptides and frg11 were derived from phospholipid-binding or fusion-related regions identified previously, it was suggested that enhancement of virus infectivity might be related to virus-cell fusion. Furthermore, enhancement was significant only when the viral peptides were pre-incubated with VHSV at the optimal low pH of fusion, before being adjusted to physiological pH and assayed for infectivity. Enhancement of VHSV infectivity caused by the pre-incubation of VHSV with peptide p5 (SAAEASAKATAEATAKG), one of the individual enhancer peptides defined from the screening of the combinatorial library, was independent of the pre-incubation pH. However, it was also related to fusion because the binding of p5 to protein G induced VHSV to bypass the endosome pathway of infection and reduced the low-pH threshold of fusion, thus suggesting an alternative virus entry pathway for p5-VHSV complexes. Further investigations into VHSV enhancer peptides might shed some light on the mechanisms of VHSV fusion.