The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting ... more The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is at present the most effective control and prevention tool, and the molecular techniques have been strongly introduced and accepted by official organizations. Among those, real time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, the droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for diagnosis and quantification of SJ and RG type VNNV, using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10 to 100-folds lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8 – 6.8 x 104 (SJ type) or 1.04 x 101 – 1.04 x 105 (RG...
The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediter... more The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56–9.88 TCID50/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID50/mL or 0.08 TCID50/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID50/mL of virus from infected tissues. The...
Con este articulo se pretende exponer de forma concisa los principales problemas patologicos, bac... more Con este articulo se pretende exponer de forma concisa los principales problemas patologicos, bacterianos y viricos, que afectan hoy en dia al cultivo de las especies de peces mas importantes, desde el punto de vista economico, en nuestro pais, haciendo especial hincapie en la efectividad de las medidas de control y prevencion empleadas normalmente en las plantas de cultivo. La informacion presentada en el mismo tiene un caracter divulgativo y esta basada en los resultados de las investigaciones llevadas a cabo en nuestro Departamento durante los ultimos anos.
Intensive fish farming at high densities results in a wide range of adverse consequences on fish ... more Intensive fish farming at high densities results in a wide range of adverse consequences on fish welfare, including pathogen spreading, stress and increased mortality rates. In this work, we have assessed whether the survival of Senegalese sole infected with the nervous necrosis virus (NNV), a pathogen responsible for severe disease outbreaks, is affected by rearing density. Based on the different fish ratios per surface area (g cm−2) and water volume (g L−1), our research showed an earlier mortality onset in the tanks containing NNV‐infected fish reared at medium density (MD: 0.071 g cm−2/5 g L−1) and high density (HD: 0.142 g cm−2/10 g L−1), as well as higher cumulative mortality values. However, transcription analysis of hsp70, gr1 and pepck genes, well‐known stress biomarkers, seems to indicate that none of the challenged fish were under high stress conditions. NNV load was slightly higher both in dead and in sampled fish from MD and HD groups, and especially in the rearing wate...
Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), ... more Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducin...
The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in f... more The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5–50 TCID50/mL), analytical spe...
The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdov... more The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the...
The susceptibility of turbot aquareovirus to five chemical agents was examined. Treatment with 5 ... more The susceptibility of turbot aquareovirus to five chemical agents was examined. Treatment with 5 mg of malachite green per liter or 500 mg of iodine per liter resulted in a 90% reduction in virus titer within 1 h. Complete inactivation within 5 min was obtained with 2% formalin, 42.5% isopropanol, or 15 mg of free available chlorine per liter. Lower concentrations of chlorine were ineffective.
In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its appl... more In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Fina...
Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among s... more Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among salmonid farmers, since losses in salmonid fish—mostly very young rainbow trout (Salmo gairdnery) fry and Atlantic salmon (Salmo salar) post-smolt—frequently reach 80–90% of stocks. The virus causing the typical signs of the IPN disease in salmonids, named infectious pancreatic necrosis virus (IPNV), has also been isolated from other fish species either suffering related diseases (then named IPNV-like virus) or asymptomatic; the general term aquabirnavirus is used to encompass all these viruses. Aquabirnaviruses are non-enveloped, icosahedral bisegmented dsRNA viruses, whose genome codifies five viral proteins, three of which are structural, and one of them is an RNA-dependent RNA polymerase. Due to the great importance of the disease, there have been great efforts to find a way to predict the level of virulence of IPNV isolates. The viral genome and proteins have been the main focus of r...
The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) ... more The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison ...
Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although viru... more Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the...
Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. ... more Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses.
Reviews: Methods and Technologies in Fish Biology and Fisheries, 2002
ABSTRACT Fifty years ago, Infectious Pancreatic Necrosis Virus (IPNV) was the first virus to be i... more ABSTRACT Fifty years ago, Infectious Pancreatic Necrosis Virus (IPNV) was the first virus to be isolated from fish, and hence the origin of the development of fish virology. After the initial studies in which farmed salmonids were considered the unique host, the virus was also found in a large number of species of freshwater and marine fishes as well as in crustaceans and molluscs. The economic impact of the disease and the ubiquity of the agent justifies the extensive literature related to the IPN virus including numerous reviews. At present, the complete genomic sequence of the dsRNA has been published for four different strains belonging to three of the serotypes described. Based on these findings, a number of molecular procedures have been developed not only to characterize the virus but also to improve the diagnostic methods, trying to circumvent the necessity for cell cultures in order to detect IPNV directly in infected fish and ova. The present review is focused on the molecular procedures used in the study of IPNV, using methods such as analysis of electropherotypes, RFLPs, nucleic acid hybridization using specific probes, and different RT-PCR protocols. The distinct procedures for extraction of viral RNA, selection of restriction enzymes, sets of primers or probes, as well as the conditions (concentration of virus, minimal time required) to obtain reliable results are examined.
Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal... more Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.
The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting ... more The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is at present the most effective control and prevention tool, and the molecular techniques have been strongly introduced and accepted by official organizations. Among those, real time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, the droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for diagnosis and quantification of SJ and RG type VNNV, using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10 to 100-folds lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8 – 6.8 x 104 (SJ type) or 1.04 x 101 – 1.04 x 105 (RG...
The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediter... more The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56–9.88 TCID50/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID50/mL or 0.08 TCID50/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID50/mL of virus from infected tissues. The...
Con este articulo se pretende exponer de forma concisa los principales problemas patologicos, bac... more Con este articulo se pretende exponer de forma concisa los principales problemas patologicos, bacterianos y viricos, que afectan hoy en dia al cultivo de las especies de peces mas importantes, desde el punto de vista economico, en nuestro pais, haciendo especial hincapie en la efectividad de las medidas de control y prevencion empleadas normalmente en las plantas de cultivo. La informacion presentada en el mismo tiene un caracter divulgativo y esta basada en los resultados de las investigaciones llevadas a cabo en nuestro Departamento durante los ultimos anos.
Intensive fish farming at high densities results in a wide range of adverse consequences on fish ... more Intensive fish farming at high densities results in a wide range of adverse consequences on fish welfare, including pathogen spreading, stress and increased mortality rates. In this work, we have assessed whether the survival of Senegalese sole infected with the nervous necrosis virus (NNV), a pathogen responsible for severe disease outbreaks, is affected by rearing density. Based on the different fish ratios per surface area (g cm−2) and water volume (g L−1), our research showed an earlier mortality onset in the tanks containing NNV‐infected fish reared at medium density (MD: 0.071 g cm−2/5 g L−1) and high density (HD: 0.142 g cm−2/10 g L−1), as well as higher cumulative mortality values. However, transcription analysis of hsp70, gr1 and pepck genes, well‐known stress biomarkers, seems to indicate that none of the challenged fish were under high stress conditions. NNV load was slightly higher both in dead and in sampled fish from MD and HD groups, and especially in the rearing wate...
Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), ... more Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducin...
The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in f... more The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5–50 TCID50/mL), analytical spe...
The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdov... more The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the...
The susceptibility of turbot aquareovirus to five chemical agents was examined. Treatment with 5 ... more The susceptibility of turbot aquareovirus to five chemical agents was examined. Treatment with 5 mg of malachite green per liter or 500 mg of iodine per liter resulted in a 90% reduction in virus titer within 1 h. Complete inactivation within 5 min was obtained with 2% formalin, 42.5% isopropanol, or 15 mg of free available chlorine per liter. Lower concentrations of chlorine were ineffective.
In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its appl... more In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Fina...
Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among s... more Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among salmonid farmers, since losses in salmonid fish—mostly very young rainbow trout (Salmo gairdnery) fry and Atlantic salmon (Salmo salar) post-smolt—frequently reach 80–90% of stocks. The virus causing the typical signs of the IPN disease in salmonids, named infectious pancreatic necrosis virus (IPNV), has also been isolated from other fish species either suffering related diseases (then named IPNV-like virus) or asymptomatic; the general term aquabirnavirus is used to encompass all these viruses. Aquabirnaviruses are non-enveloped, icosahedral bisegmented dsRNA viruses, whose genome codifies five viral proteins, three of which are structural, and one of them is an RNA-dependent RNA polymerase. Due to the great importance of the disease, there have been great efforts to find a way to predict the level of virulence of IPNV isolates. The viral genome and proteins have been the main focus of r...
The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) ... more The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison ...
Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although viru... more Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the...
Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. ... more Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses.
Reviews: Methods and Technologies in Fish Biology and Fisheries, 2002
ABSTRACT Fifty years ago, Infectious Pancreatic Necrosis Virus (IPNV) was the first virus to be i... more ABSTRACT Fifty years ago, Infectious Pancreatic Necrosis Virus (IPNV) was the first virus to be isolated from fish, and hence the origin of the development of fish virology. After the initial studies in which farmed salmonids were considered the unique host, the virus was also found in a large number of species of freshwater and marine fishes as well as in crustaceans and molluscs. The economic impact of the disease and the ubiquity of the agent justifies the extensive literature related to the IPN virus including numerous reviews. At present, the complete genomic sequence of the dsRNA has been published for four different strains belonging to three of the serotypes described. Based on these findings, a number of molecular procedures have been developed not only to characterize the virus but also to improve the diagnostic methods, trying to circumvent the necessity for cell cultures in order to detect IPNV directly in infected fish and ova. The present review is focused on the molecular procedures used in the study of IPNV, using methods such as analysis of electropherotypes, RFLPs, nucleic acid hybridization using specific probes, and different RT-PCR protocols. The distinct procedures for extraction of viral RNA, selection of restriction enzymes, sets of primers or probes, as well as the conditions (concentration of virus, minimal time required) to obtain reliable results are examined.
Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal... more Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.
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Papers by Carlos Dopàzo