Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I ifn.

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.

Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles.

Virus like particles (VLPs) against viral pathogens not only constitute a novel approach for the development of antiviral vaccines for an specific virus, but also for the creation of multivalent vaccines in which antigens from other pathogens may be expressed on the surface of these VLPs. Despite positive results on protection for many of these VLPs in both fish and mammals, not many studies have focused on the immune response triggered by these particles; studies that may provide hints for the identification of immune mechanisms responsible for antiviral protection, which are mostly unknown in fish. In the current work, we have studied the levels of transcription of several immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) intraperitoneally injected with VLPs from infectious pancreatic necrosis virus (IPNV) focusing on the chemokine response as well as the response of genes related to interferon (IFN) production. Surprisingly, the capacity of VLPs to induce chemokines differed from that of live IPNV, suggesting a direct effect of viral replication on the chemokine response in this organ. While VLPs up-regulated the transcription of CK3, CK10 and CXCd and down-modulated CK5B, CK6 and CK9 transcription, a previous study in which the transcription of γIP, CXCd, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12 had been studied demonstrated that IPNV only significantly up-regulated CK6 and down-modulated CK3 in the spleen. On the other hand, the administration of VLPs produced a strong mobilization to the peritoneum of CD4(+), IgM(+), IgT(+) and CD83(+) leukocytes similar to that induced by the live viral infection. In both cases, this leukocyte recruitment seemed to be greatly mediated through CK3, CK5B, CK9 and CK10 chemokine production. These results together with the fact that VLPs strongly induced non-specific lymphocyte proliferation and specific anti-IPNV antibody production point to VLPs as excellent candidates for vaccine development.

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

Salmonid fish viruses and cell interactions at early steps of the infective cycle.

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.

Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures.

An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10(3) TCID50/ml, whilst the labelling appears at 24h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent.

Virulence of infectious hematopoietic necrosis virus and Infectious pancreatic necrosis virus coinfection in rainbow trout ( Oncorhynchus mykiss) and nucleotide sequence analysis of the IHNV glycoprotein gene.

The outcomes of a coinfection of rainbow trout ( Oncorhynchus mykiss) with Infectious hematopoietic necrosis virus (IHNV) strain S46 and Infectious pancreatic necrosis virus (IPNV) strain S46 was determined after waterborne infection. Trout infected with the IHNV/IPNV.S46 sample, (a mixed sample containing equal infectious titers of the viruses) showed 50% less mortality than fish infected with either of the reference viruses alone. Forty-five days after the coinfection, IPNV antigens were detected by flow cytometry in 49 to 63% of the leukocytes from the surviving trout; whereas, only 9-15.6% of the leukocytes expressed IHNV viral antigens. IPNV was easily detected by reverse transcription-polymerase chain reaction (RT-PCR), whereas, for IHNV, a second step of amplification of a 753 bp fragment corresponding to the internal sequences of the IHNV G gene was necessary to optimize viral detection. The sequence of the IHNV gene involved in virulence, the glycoprotein (G) gene, was determined for the IHNV.S46 and compared with other sequences available in the GenBank. Changes found were not located in the antigenic domains of the glycoprotein and were considered not significant.

Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish.

In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1x10(2) and 1x10(3) TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.

Antigenic properties and experimental transmission to several fish species of a marine birnavirus isolated from sole (Solea senegalensis).

A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the solevirus to other fish species has been determined by inoculation to freshwater and marine fish species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the marine fish species, although solevirus caused an asymptomatic infection in all species tested, as demonstrated by the detection of viral RNA and of viral antigens in fish leucocytes, respectively, using polymerase chain reaction (PCR) and flow cytometry (FC).

Nested PCR improves detection of infectious hematopoietic necrosis virus in cells coinfected with infectious pancreatic necrosis virus.

A nested assay using the reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures coinfected with infectious pancreatic necrosis virus (IPNV). Two pairs of primers were designed: one for the amplification of glycoprotein G-specific gene RNA from IHNV (or 1512 bp fragment), and the other for the amplification of an inner 753 bp fragment using the cDNA from the G gene as substrate. Direct RT-PCR was also developed for the amplification of a VP-2 gene fragment from IPNV (613 bp fragment); this method always detected the virus IPNV in the coinfected cells tested but the amplification of IHNV was not as readily achieved. IHNV, however, was detected specifically by nested PCR in coinfected cells at a multiplicity of infection that was 1000 times lower than that of IPNV. Nested PCR was therefore more sensitive than direct RT-PCR for IHNV, and may thus be more appropriate for the detection of low infective titers of IHNV in the presence of IPNV when interference occurs.

Viral coinfection in salmonids: infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus.

Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log10 units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48-72 h post infection, in contrast to the 50-80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections.

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream.

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.

Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, sialidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabream, Sparus aurata. Variable amounts of glycoproteins containing sialic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, mannose and/or glucose residues were observed in the cuticle and mucous cells of the corporal skin, tails and fins. Germinative and epithelial cells of the epidermis contained glycogen, proteins, carboxylated groups, as well as glycoproteins with mannose and/or glucose and N-acetyl-D-galactosamine residues. Hyaline capsule of the mature lymphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1). Con A reacted with the granular cytoplasm, specially around hyaline capsule, and with the basophilic intracytoplasmic inclusions developed in mature lymphocystis-infected cells of Sparus aurata skin. These sugar residues (mannose and/or glucose), as well as N-acetyl-D-glucosamine and/or sialic acid and N-acetyl-D-galactosamine were not detected in the hyaline capsule of the lymphocystis disease.

Antiviral activity of a D-glucosamine derivative against herpetic ulcers (HSV type 2) in rabbit cornea.

Although most herpetic ocular infections in adults are caused by herpesvirus hominis type 1, several cases of culture proved HSV-2 ocular infection in adults have been described, with more severe and prolonged disease. In a screening for new antiherpetic compounds, we investigated the efficacy in vivo of a new compound, nitroderivative of D-glucosaminhydrochloride (GN-11) in comparison with D-glucosaminhydrochloride (GN), Acyclovir (ACV) and placebo against herpetic keratitis of herpes simplex type 2 in 4 x 4 eyes from 4 x 4 rabbits, respectively. ACV and GN-11 showed similar results. The treatment with GN-11 retarded the appearance of herpetic lesions, which were small and diffuse in comparison with the placebo group. A total recovery was obtained on the 12th day of the treatment. In the ACV treated group, a minimal number of small lesions appeared, but the eyes recovered normality on the 7th day of treatment. The appearance of acute herpetic keratitis was prevented by GN-11. Placebo and GN treated groups showed similar evolution, with lost vision and neurological involvement on the 7th day of infection.

Suppression of persistent varicella-zoster virus infection in Vero cells by acyclovir.

Persistent infection was established in Vero cells inoculated with varicella-zoster virus (VZV)-infected WI-38 cells. Treatment with 9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) at doses of 100, 80, 40, and 10 micrograms/ml eliminated the infectious virus, lower doses such as 0.1 and 0.01 micrograms/ml were ineffective.