Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells.

The understanding of virus-host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection.

Synergistic effects in the antiviral activity of the three Mx proteins from gilthead seabream (Sparus aurata).

Due to their direct antiviral activity, Mx proteins play a main role in the response mediated by type I interferon against viral infections. The study on gilthead seabream Mx proteins is especially interesting, since this species is unusually resistant to viral diseases, being asymptomatic carrier of several viruses pathogenic to other fish species. Gilthead seabream has three Mx proteins (Mx1, Mx2 and Mx3) that, separately, display antiviral activity against a wide range of viruses, showing interesting differences in their antiviral specificities. In this work, the possible synergy between the three Mx isoforms has been studied using in vitro systems consisting of CHSE-214 cells stably expressing two or the three gilthead seabream Mx proteins. The antiviral activity of these Mx combinations has been tested against the Infectious Pancreatic Necrosis Virus (IPNV), the Viral Haemorrhagic Septicaemia Virus (VHSV), the European Sheatfish Virus (ESV) and the Lymphocystis Disease Virus (LCDV). A synergistic effect of the Mx proteins was only detected against ESV, no synergy was observed against LCDV, and a negative interference was detected against the two RNA viruses tested, IPNV and VHSV, as viral replication was higher in cells expressing certain Mx combinations than in cells expressing these proteins separately. These results suggest a functional interaction between gilthead seabream Mx isoforms, which results in a higher or lower antiviral activity depending on the virus tested, thus supporting the idea of complex virus-host interactions and finely tuned mechanisms controlling the antiviral activity of Mx proteins.

Experimental susceptibility of European sea bass and Senegalese sole to different betanodavirus isolates.

The susceptibility of juvenile European sea bass and Senegalese sole to three VNNV isolates (a reassortant RGNNV/SJNNV, as well as the parental RGNNV and SJNNV genotypes) has been evaluated by challenges using two inoculation ways (bath and intramuscular injection). The results demonstrate that these two fish species are susceptible to all the VNNV isolates tested. In European sea bass, RGNNV caused the highest cumulative mortality, reaching maximum values of viral RNA and titres. Although the SJNNV isolate did not provoke mortality or clinical signs of disease in this fish species, viral production in survivor fish was determined; on the other hand the reassortant isolate did cause mortality and clinical signs of disease, although less evident than those recorded after RGNNV infection. These results suggest that the changes suffered by the SJNNV RNA2 segment of the reassortant isolate, compared to the parental SJNNV, may have involved host-specificity and/or virulence determinants for European sea bass. Regarding Senegalese sole, although the three isolates caused 100% mortality, the reassortant strain provoked the most acute symptoms, and more quickly, especially in the bath challenge. This was also the isolate showing less difference between the number of RNA copies and viral titre, reaching the highest titres of infective viral particles in nervous tissue of infected animals. The RGNNV isolate produced the lowest values of infective viral particles. All these results suggest that the RGNNV and the reassortant isolates are the most suited for infecting European sea bass and Senegalese sole, respectively.

Mx1, Mx2 and Mx3 proteins from the gilthead seabream (Sparus aurata) show in vitro antiviral activity against RNA and DNA viruses.

Mx proteins are important components of the antiviral innate immune response mediated by type I interferon. Classically, these proteins have been considered to be triggered by viral RNA, thus showing activity against RNA viruses. Actually, three Mx proteins (SauMx1, SauMx2 and SauMx3) from gilthead seabream (Sparus aurata) have previously shown antiviral activity against a dsRNA virus: the infectious pancreatic necrosis virus (IPNV) in vitro. For further characterizing their antiviral spectrum, the activity of SauMx proteins were tested against three different viral pathogens of fish: the lymphocystis disease virus (LCDV, a dsDNA virus), a pathogen of gilthead seabream; the viral haemorrhagic septicaemia virus (VHSV, a ssRNA virus), to which gilthead seabream is considered a reservoir species; and the European sheatfish virus (ESV, a dsDNA virus), that has not been detected in gilthead seabream to date. Three clonal populations of CHSE-214 cells developed in a previous study, stably expressing SauMx1, SauMx2 and SauMx3, respectively, were challenged with the three viruses. Results combining cytopathic effects and virus yield reduction assays showed that SauMx1 protected the cells against VHSV and LCDV, SauMx2 protected against ESV and LCDV, and SauMx3 showed activity only against VHSV. This study, besides confirming the antiviral activity of the three gilthead seabream Mx proteins, is the first report of the protective effect of a fish Mx against DNA viruses. Additionally, it discloses a clear specificity between Mx proteins and virus targets, supporting the idea that the relationship between virus and Mx proteins is finely tuned.

Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae.

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR-hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF-1 cells. Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV-positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR-hybridization) or in larvae (PCR-hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR-hybridization, ISH and IHC. After feeding on LCDV-positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.

Differential antiviral activity of Mx1, Mx2 and Mx3 proteins from gilthead seabream (Sparus aurata) against Infectious Pancreatic Necrosis Virus (IPNV).

Mx proteins are crucial effectors of the innate antiviral response mediated by the interferon type I signalling pathway. Recently, three Mx proteins, named SauMx1, SauMx2 and SauMx3, corresponding to three different genes, have been identified in the cultured marine species gilthead seabream (Sparus aurata). In this study, the three SauMx cDNAs were cloned into expression vectors and used for transfection of CHSE-214 cells. Monoclonal cell populations stably expressing each recombinant protein have been obtained and characterized. The protection conferred by each recombinant SauMx against Infectious Pancreatic Necrosis Virus (IPNV) infection has been in vitro evaluated, having found clear differences among them. According to the cytopathic effects and the virus yield reduction assays, only cells expressing SauMx2 and SauMx3 showed significant resistance to IPNV infection. Otherwise, quantitative RT real-time PCR assays suggested that each SauMx protein has a different target during the viral inhibition process. The differences observed among the three SauMx proteins are discussed in terms of their differential mechanism of action and antiviral specificity, suggesting, as a whole, to play a synergistic activity in the protection of gilthead seabream against IPNV.

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

AIMS: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. METHODS AND RESULTS: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). CONCLUSIONS: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream, Pagrus auriga Valenciennes, and common seabream, Pagrus pagrus (L.), using a non-destructive procedure.

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.

Poly I:C induces Mx transcription and promotes an antiviral state against sole aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup).

Mx is an interferon-induced protein that protects against viral infections. In this study the absolute number of Mx transcripts after poly I:C injection (a synthetic dsRNA) or sole aquabirnavirus (solevirus) inoculation in Senegalese sole (Solea senegalensis Kaup) has been quantified. Mx expression profiles differed clearly in both experimental conditions; the induction response was faster and more intense after poly I:C injection than after solevirus inoculation. Moreover, pre-injection of soles with poly I:C prior to solevirus infection eliminated the induction of Mx expression associated with this virus. To evaluate the possible interference of poly I:C treatments on solevirus replication, the mRNA levels of the virus capsid protein (VP2) were determined by RT-PCR. VP2 transcripts were hardly detected in poly I:C pre-injected animals from 12 to 72 h after solevirus inoculation. All these data suggest that poly I:C is able to induce an antiviral state that interferes with solevirus replication, and support the suitability of Mx expression analysis as a marker to study the defensive response against solevirus.

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species.

AIMS: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. METHODS AND RESULTS: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). CONCLUSIONS: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.

Microbiological quality of reclaimed water used for golf courses' irrigation.

Microbial quality of reclaimed water used for irrigation in two golf courses located in the southern Iberian Peninsula (Spain and Portugal) was evaluated. Bacterial indicators for faecal pollution (total and faecal coliforms, Escherichia coli and enterococci) were tested by membrane filtration using appropriate selective media. In addition, somatic E. coli bacteriophages, enteric viruses (entero-, hepatitis A and rota-) and Legionella pneumophila were also analysed. The results obtained showed that all wastewater treatment processes reduced adequately the number of indicator microorganisms although a significant correlation between pathogenic and indicator microorganisms tested was not found. L. pneumophila was detected by PCR but not confirmed by culture. Survival experiments of pathogenic microorganisms in aerosols and irrigated turf are conducted to determine the health hazards for the golf practice and to propose a microbial standard for wastewater used for irrigation of golf courses.

Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique.

An immunoblot technique for the detection of lymphocystis disease virus (LCDV) in naturally infected gilt-head seabream (Sparus aurata, L.) has been developed. A specific antiserum against a 60 kDa viral protein has been proven to be an appropriate tool for LCDV diagnosis either from inoculated cell cultures or from fish tissues using the immunoblot assay. The sensitivity of this technique varied between 10(-1) and 10(2) TCID50. LCDV has also been detected in fish tissues from both, diseased and asymptomatic gilt-head seabream. For the asymptomatic fish detection, a viral amplification step in cell culture and a subsequent viral concentration using polyethylene glycol (PEG) (600 wt) are required. On the contrary, immunoblot allowed the detection of LCDV antigens directly from tissue homogenates of diseased fish. The method described in this study shows higher sensitivity than classical detection techniques based on cell culture inoculation.

Isolation of lymphocystis disease virus from sole, Solea senegalensis Kaup, and blackspot sea bream, Pagellus bogaraveo (Brunnich).

Two viruses were isolated from cultured sole, Solea senegalensis, and wild blackspot sea bream, Pagellus bogaraveo, and preliminarily characterized as lymphocystis disease viruses (LCDVs). Viral isolates were characterized by morphological, biochemical and biophysical properties. In addition, the susceptibility of four fish cell lines was also tested. LCDV isolates developed cytopathic effects on the SAF-1 cell line at 5 and 6 days post-infection and reached titres of 10(6) TCID50 mL(-1). The antigenic and structural protein analysis of the two new LCDV isolates showed identical profiles to that obtained for LCDV strain Leetown NFH (ATCC VR-342), used as a reference viral strain, and for an LCDV isolate collected from gilt-head sea bream, Sparus aurata, cultured in southern Spain. Molecular confirmation was performed by polymerase chain reaction. Specific primers for LCDV produced a 270-bp DNA fragment, the expected size for LCDV.

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).

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.