Suppression subtractive hybridization was used to identify differentially expressed genes in hemo... more Suppression subtractive hybridization was used to identify differentially expressed genes in hemocytes from carpet-shell clam Ruditapes decussatus stimulated with a mixture of dead bacterial strains. Putative function could be assigned to 100 of the 253 sequenced cDNAs. Based on sequence homologies, 3.16% of the total identified genes were possibly related to immune functions. Clam myticin isoforms 1, 2 and 3,
... et al., 1979 DP Anderson, B. Roberson and OW Dixon, Cellular response in rainbow trout, Salmo... more ... et al., 1979 DP Anderson, B. Roberson and OW Dixon, Cellular response in rainbow trout, Salmo gairdnieri Richardson to Yersinia ... Figueras et al., 1997 A. Figueras, MM Santarém and B. Novoa, In vitro immunostimulation of turbot (Scophthalmus maximus) leucocytes with β ...
Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditap... more Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host-pathogen interactions in R. decussatus.
Interferons play a significant role in host resistance to viral infections. Mx proteins induced b... more Interferons play a significant role in host resistance to viral infections. Mx proteins induced by type I IFN are one of the best studied determinants of innate immunity, although the mechanisms of the antiviral actions of these proteins have not been completely clarified [1]. Mx genes have been cloned and characterised in mammals, birds and fish [2e8], and the antiviral activity of Mx proteins has been established for mouse, rat, human, chicken and, very recently, flounder and Atlantic salmon [9e13]. Fish Mx genes are induced by IFN and double-stranded RNA (dsRNA) polyinosinic:polycytidylic acid (poly I:C), which is a well-known inducer of type I IFN both in mammals [14,15] and fish [16e20]. Recently, Lutfalla et al. [19] have identified seven Mx genes in zebrafish (zMxA to zMxG) and have found evidence of expression for all of them except zMxF. Moreover, three full-length Mx cDNAs have been cloned and sequenced from Atlantic salmon [21], rainbow trout [7,22] and goldfish [23], and one Mx cDNA from perch [6], Atlantic halibut [8], Japanese flounder [24], channel catfish (AY095349), gilthead sea bream [25], pufferfish [19,26], Chinese perch (AY392097) and European flounder (AJ606083). The understanding of the role of Mx proteins in cultured fish species is not only important to elucidate the antiviral mechanisms of these proteins but also to use up-regulated expression of the Mx gene as a molecular marker for type I IFN induction. The present work describes the cloning and characterisation of a full-length Mx cDNA from turbot. Expression of this gene in different organs and blood leucocytes was induced by poly I:C, in order to evaluate its capacity to regulate the Mx gene both in vivo and in vitro.
The alternative pathway is considered to be the most ancient route for activation of the compleme... more The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.
Several non-specific immune responses of turbot (Scophthalmus maximusL.) were enhanced after the ... more Several non-specific immune responses of turbot (Scophthalmus maximusL.) were enhanced after the administration ofβ-glucans and/or O-antigen ofVibrio damsela. Bactericidal activity of turbot leucocytes varied depending on the bacterial virulence. Whilst no differences in bactericidal activity were observed among treated and untreated fish when a virulent strain ofV. damselawas used, leucocytes from glucan plus O-antigen-treated turbot efficiently destroyed an avirulentV. damselastrain.
There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here,... more There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here, we report the use of poly(I:C) stabilized with chitosan as an adjuvant for development of better finfish vaccines. The adjuvant was co-injected with inactivated viral hemorrhagic septicemia virus (VHSV) (CSpIC+iV vaccine) in adult zebrafish and its efficiency in protection against VHSV infection was compared to a live, attenuated VHS virus vaccine (aV). Both free and stabilized poly(I:C) were strong inducers of an antiviral state, measured by transcriptional activation of the genes of viral sensors: toll-like receptors, interferons, and interferon-stimulated genes, such as MXa within 48 h after injection. Both the CSpIC+iV and the aV formulations provided a significant protection against VHSV-induced mortality. However, when plasma from survivors was tested for neutralizing antibodies in an in vitro protection assay, we could not demonstrate any protective effect. On the contrary, plasm...
Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible s... more Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the &quo...
Viral haemorrhagic septicaemia virus (VHSV), a well known salmonids pathogen, has also been repor... more Viral haemorrhagic septicaemia virus (VHSV), a well known salmonids pathogen, has also been reported to be pathogenic for turbot (Scophthalmus maximus). In the present work, the replication of VHSV was studied in vitro in turbot head kidney macrophages and blood leukocytes. VHSV was able to infect both primary cultures and viral titer increased with time, either inside the cells or in the supernatant. However, no cytopathic effect was observed during the experiments and the titers were always lower than those obtained in the fish cell lines. The number of trout and turbot macrophages after several days of in vitro infection with VHSV was compared with uninfected controls by viable cell count but no significant differences were observed. The number of cells supporting viral replication evaluated by immunofluorescence in trout and turbot was low (8 and 1.7%, respectively). Respiratory burst activity of head kidney macrophages was assayed at different days post-infection, but no significant differences were found between the control and the infected cultures neither in trout nor turbot.
In the present work, we have studied the role of nitric oxide (NO) on the replication of viral ha... more In the present work, we have studied the role of nitric oxide (NO) on the replication of viral haemorrhagic septicemia virus (VHSV), a virus which produces high mortalities in fish aquaculture worldwide and that is known to replicate in turbot (Scophthalmus maximus) head kidney macrophages. Viral infection of turbot kidney macrophages in vitro induced an up-regulation of NO production and we have tested whether this endogenous NO production induced by VHSV on macrophages had an antiviral effect using the NO synthase inhibitor, N-omega-nitro-L-arginine (L-NAME). When L-NAME was added to the VHSV-infected cultures, no increase on VHSV titer was observed, even though the inhibitor was capable of decreasing NO production. When exogenous NO was apported by the nitric oxide donor, glycerin trinitrate (GTN) an antiviral effect on VHSV was observed. The NO donor significantly inhibited VHSV replication on a turbot fibroblast cell line (TV-1) and on turbot kidney macrophages.
The rhabdovirus viral haemorrhagic septicemia virus (VHSV) is the etiological agent of one of the... more The rhabdovirus viral haemorrhagic septicemia virus (VHSV) is the etiological agent of one of the most important salmonid viral diseases. In the present work, the ability of VHSV to infect and replicate in zebrafish at low temperature (15 degrees C) was demonstrated. Zebrafish was also used to determine the effectiveness of the recombinant virus rIHNV-Gvhsv GFP as a live attenuated vaccine against the virulent VHSV strain. Fish intraperitoneally injected with 3 x 10(6) to 3 x 10(5)TCID50/ml of the wild type VHSV showed a 100% of cumulative mortality, meanwhile only 57% of mortality was obtained in bath infections. Infected fish showed external clinical signs and histological observations revealed the appearance of small haemorrhages in the muscle, kidney, liver and dermis. Neither mortalities nor clinical signs were recorded in fish infected with a live attenuated recombinant virus. By RT-PCR technique, VHSV was detected in all the organs as early as 24h, but the recombinant virus was not detected in all the sampled days. VHSV was able to replicate "in vitro" in head kidney cells but the replication capacity of the attenuated viral strain was limited. The recombinant virus rIHNV-Gvhsv GFP was able to protect against VHSV with a survival rate ranging from 20% to 60% depending of the vaccine dose. The increase of TLR3, IFNalphabeta, Mx, IFNgamma and TNFalpha expression at 72h post-infection in the kidney of VHSV-infected fish contrasted with the results obtained with the avirulent virus, which did not induce an increment of this expression in infected fish. Zebrafish is a suitable animal model to study VHSV infection and immune (innate and adaptive) responses and, more importantly, we demonstrate for the first time the usefulness of the zebrafish as a vaccination model to viral diseases. In addition, the high protection obtained with the live attenuated virus demonstrates that the zebrafish is able to mount an efficient antiviral immune response at 15 degrees C.
Suppression subtractive hybridization was used to identify differentially expressed genes in hemo... more Suppression subtractive hybridization was used to identify differentially expressed genes in hemocytes from carpet-shell clam Ruditapes decussatus stimulated with a mixture of dead bacterial strains. Putative function could be assigned to 100 of the 253 sequenced cDNAs. Based on sequence homologies, 3.16% of the total identified genes were possibly related to immune functions. Clam myticin isoforms 1, 2 and 3,
... et al., 1979 DP Anderson, B. Roberson and OW Dixon, Cellular response in rainbow trout, Salmo... more ... et al., 1979 DP Anderson, B. Roberson and OW Dixon, Cellular response in rainbow trout, Salmo gairdnieri Richardson to Yersinia ... Figueras et al., 1997 A. Figueras, MM Santarém and B. Novoa, In vitro immunostimulation of turbot (Scophthalmus maximus) leucocytes with β ...
Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditap... more Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host-pathogen interactions in R. decussatus.
Interferons play a significant role in host resistance to viral infections. Mx proteins induced b... more Interferons play a significant role in host resistance to viral infections. Mx proteins induced by type I IFN are one of the best studied determinants of innate immunity, although the mechanisms of the antiviral actions of these proteins have not been completely clarified [1]. Mx genes have been cloned and characterised in mammals, birds and fish [2e8], and the antiviral activity of Mx proteins has been established for mouse, rat, human, chicken and, very recently, flounder and Atlantic salmon [9e13]. Fish Mx genes are induced by IFN and double-stranded RNA (dsRNA) polyinosinic:polycytidylic acid (poly I:C), which is a well-known inducer of type I IFN both in mammals [14,15] and fish [16e20]. Recently, Lutfalla et al. [19] have identified seven Mx genes in zebrafish (zMxA to zMxG) and have found evidence of expression for all of them except zMxF. Moreover, three full-length Mx cDNAs have been cloned and sequenced from Atlantic salmon [21], rainbow trout [7,22] and goldfish [23], and one Mx cDNA from perch [6], Atlantic halibut [8], Japanese flounder [24], channel catfish (AY095349), gilthead sea bream [25], pufferfish [19,26], Chinese perch (AY392097) and European flounder (AJ606083). The understanding of the role of Mx proteins in cultured fish species is not only important to elucidate the antiviral mechanisms of these proteins but also to use up-regulated expression of the Mx gene as a molecular marker for type I IFN induction. The present work describes the cloning and characterisation of a full-length Mx cDNA from turbot. Expression of this gene in different organs and blood leucocytes was induced by poly I:C, in order to evaluate its capacity to regulate the Mx gene both in vivo and in vitro.
The alternative pathway is considered to be the most ancient route for activation of the compleme... more The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.
Several non-specific immune responses of turbot (Scophthalmus maximusL.) were enhanced after the ... more Several non-specific immune responses of turbot (Scophthalmus maximusL.) were enhanced after the administration ofβ-glucans and/or O-antigen ofVibrio damsela. Bactericidal activity of turbot leucocytes varied depending on the bacterial virulence. Whilst no differences in bactericidal activity were observed among treated and untreated fish when a virulent strain ofV. damselawas used, leucocytes from glucan plus O-antigen-treated turbot efficiently destroyed an avirulentV. damselastrain.
There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here,... more There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here, we report the use of poly(I:C) stabilized with chitosan as an adjuvant for development of better finfish vaccines. The adjuvant was co-injected with inactivated viral hemorrhagic septicemia virus (VHSV) (CSpIC+iV vaccine) in adult zebrafish and its efficiency in protection against VHSV infection was compared to a live, attenuated VHS virus vaccine (aV). Both free and stabilized poly(I:C) were strong inducers of an antiviral state, measured by transcriptional activation of the genes of viral sensors: toll-like receptors, interferons, and interferon-stimulated genes, such as MXa within 48 h after injection. Both the CSpIC+iV and the aV formulations provided a significant protection against VHSV-induced mortality. However, when plasma from survivors was tested for neutralizing antibodies in an in vitro protection assay, we could not demonstrate any protective effect. On the contrary, plasm...
Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible s... more Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the &quo...
Viral haemorrhagic septicaemia virus (VHSV), a well known salmonids pathogen, has also been repor... more Viral haemorrhagic septicaemia virus (VHSV), a well known salmonids pathogen, has also been reported to be pathogenic for turbot (Scophthalmus maximus). In the present work, the replication of VHSV was studied in vitro in turbot head kidney macrophages and blood leukocytes. VHSV was able to infect both primary cultures and viral titer increased with time, either inside the cells or in the supernatant. However, no cytopathic effect was observed during the experiments and the titers were always lower than those obtained in the fish cell lines. The number of trout and turbot macrophages after several days of in vitro infection with VHSV was compared with uninfected controls by viable cell count but no significant differences were observed. The number of cells supporting viral replication evaluated by immunofluorescence in trout and turbot was low (8 and 1.7%, respectively). Respiratory burst activity of head kidney macrophages was assayed at different days post-infection, but no significant differences were found between the control and the infected cultures neither in trout nor turbot.
In the present work, we have studied the role of nitric oxide (NO) on the replication of viral ha... more In the present work, we have studied the role of nitric oxide (NO) on the replication of viral haemorrhagic septicemia virus (VHSV), a virus which produces high mortalities in fish aquaculture worldwide and that is known to replicate in turbot (Scophthalmus maximus) head kidney macrophages. Viral infection of turbot kidney macrophages in vitro induced an up-regulation of NO production and we have tested whether this endogenous NO production induced by VHSV on macrophages had an antiviral effect using the NO synthase inhibitor, N-omega-nitro-L-arginine (L-NAME). When L-NAME was added to the VHSV-infected cultures, no increase on VHSV titer was observed, even though the inhibitor was capable of decreasing NO production. When exogenous NO was apported by the nitric oxide donor, glycerin trinitrate (GTN) an antiviral effect on VHSV was observed. The NO donor significantly inhibited VHSV replication on a turbot fibroblast cell line (TV-1) and on turbot kidney macrophages.
The rhabdovirus viral haemorrhagic septicemia virus (VHSV) is the etiological agent of one of the... more The rhabdovirus viral haemorrhagic septicemia virus (VHSV) is the etiological agent of one of the most important salmonid viral diseases. In the present work, the ability of VHSV to infect and replicate in zebrafish at low temperature (15 degrees C) was demonstrated. Zebrafish was also used to determine the effectiveness of the recombinant virus rIHNV-Gvhsv GFP as a live attenuated vaccine against the virulent VHSV strain. Fish intraperitoneally injected with 3 x 10(6) to 3 x 10(5)TCID50/ml of the wild type VHSV showed a 100% of cumulative mortality, meanwhile only 57% of mortality was obtained in bath infections. Infected fish showed external clinical signs and histological observations revealed the appearance of small haemorrhages in the muscle, kidney, liver and dermis. Neither mortalities nor clinical signs were recorded in fish infected with a live attenuated recombinant virus. By RT-PCR technique, VHSV was detected in all the organs as early as 24h, but the recombinant virus was not detected in all the sampled days. VHSV was able to replicate "in vitro" in head kidney cells but the replication capacity of the attenuated viral strain was limited. The recombinant virus rIHNV-Gvhsv GFP was able to protect against VHSV with a survival rate ranging from 20% to 60% depending of the vaccine dose. The increase of TLR3, IFNalphabeta, Mx, IFNgamma and TNFalpha expression at 72h post-infection in the kidney of VHSV-infected fish contrasted with the results obtained with the avirulent virus, which did not induce an increment of this expression in infected fish. Zebrafish is a suitable animal model to study VHSV infection and immune (innate and adaptive) responses and, more importantly, we demonstrate for the first time the usefulness of the zebrafish as a vaccination model to viral diseases. In addition, the high protection obtained with the live attenuated virus demonstrates that the zebrafish is able to mount an efficient antiviral immune response at 15 degrees C.
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