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Frederick S. B. Kibenge - One of the best experts on this subject based on the ideXlab platform.
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology Journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus , family Orthomyxoviridae . ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Results Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. Conclusions This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of european and north american genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcri
Virology Journal, 2010Co-Authors: Vikas Kulshreshtha, Molly J. T. Kibenge, Kira Salonius, Nathalie Simard, Angela Riveroll, Frederick S. B. KibengeAbstract:Background Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon ( Salmo salar ); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae , genus, Isavirus . The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts. Results Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%. Conclusions We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CA^T/_ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.
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Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of European and North American genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcri
Virology Journal, 2010Co-Authors: Vikas Kulshreshtha, Molly J. T. Kibenge, Kira Salonius, Nathalie Simard, Angela Riveroll, Frederick S. B. KibengeAbstract:Background Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts.
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Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences
Virology journal, 2009Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Yingwei Wang, Marcos Godoy, Valentina Gherardelli, Soledad Mansilla, Angelica Lisperger, Miguel Jarpa, Geraldine Larroquete, Fernando AvendañoAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparision between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV.
Molly J. T. Kibenge - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH Open Access Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype
2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Ra Morton, Richard RoutledgeAbstract:Background: Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan ® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods: In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PC
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology Journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus , family Orthomyxoviridae . ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Results Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. Conclusions This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Infectious salmon anaemia virus (ISAV) in Chilean Atlantic salmon (Salmo salar) aquaculture: emergence of low pathogenic ISAV-HPR0 and re-emergence of virulent ISAV-HPR∆: HPR3 and HPR14
Virology Journal, 2013Co-Authors: Marcos G Godoy, Molly J. T. Kibenge, Rudy Suarez, Eduardo Lazo, Alejandro Heisinger, Javier Aguinaga, Diego Bravo, Julio Mendoza, Katerina O Llegues, Rubén Avendaño-herreraAbstract:Infectious salmon anaemia (ISA) is a serious disease of marine-farmed Atlantic salmon ( Salmo salar ) caused by ISA virus (ISAV), which belongs to the genus Isavirus , family Orthomyxoviridae . ISA is caused by virulent ISAV strains with deletions in a highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein (designated virulent ISAV-HPR∆). This study shows the historic dynamics of ISAV-HPR∆ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPR∆ in Chile, and reports the 2013 ISA outbreak in Chile. The first ISA outbreak in Chile occurred from mid-June 2007 to 2010 and involved the virulent ISAV-HPR7b, which was then replaced by a low pathogenic ISAV-HPR0 variant. We analyzed this variant in 66 laboratory-confirmed ISAV-HPR0 cases in Chile in comparison to virulent ISAV-HPR∆ that caused two new ISA outbreaks in April 2013. Multiple alignment and phylogenetic analysis of HE sequences from all ISAV-HPR0 viruses allowed us to identify three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 (^360PST^362, ^360PAN^362 and ^360PAT^362) in HPR. The virus responsible for the 2013 ISAV-HPR∆ cases in Chile belonged to ISAV-HPR3 and ISAV-HPR14, and in phylogenetic analyses, both clustered with the ISAV-HPR0 found in Chile. The ISAV-HPR14 had the ISAV-HPR0 residue pattern ^360PAT^362, which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPR∆ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPR∆ to Chile. The clinical presentations and diagnostic evidence of the 2013 ISA cases indicated a mixed infection of ISAV with the ectoparasite Caligus rogercresseyi and the bacterium Piscirickettsia salmonis , which underscores the need for active ISAV surveillance in areas where ISAV-HPR0 is enzootic, to ensure early detection and control of new ISA outbreaks, as it is considered a risk factor. This is the first report of ISA linked directly to the presence of ISAV-HPR0, and provides strong evidence supporting the contention that ISAV-HPR0 shows a strong relationship to virulent ISAV-HPR∆ viruses and the possibility that it could mutate to virulent ISAV-HPR∆.
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Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of european and north american genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcri
Virology Journal, 2010Co-Authors: Vikas Kulshreshtha, Molly J. T. Kibenge, Kira Salonius, Nathalie Simard, Angela Riveroll, Frederick S. B. KibengeAbstract:Background Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon ( Salmo salar ); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae , genus, Isavirus . The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts. Results Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%. Conclusions We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CA^T/_ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.
Yingwei Wang - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH Open Access Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype
2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Ra Morton, Richard RoutledgeAbstract:Background: Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan ® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods: In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PC
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology Journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus , family Orthomyxoviridae . ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Results Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. Conclusions This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
Virology journal, 2016Co-Authors: Molly J. T. Kibenge, Yingwei Wang, Tokinori Iwamoto, Richard Routledge, Alexandra Morton, Frederick S. B. KibengeAbstract:Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
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Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences
Virology journal, 2009Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Yingwei Wang, Marcos Godoy, Valentina Gherardelli, Soledad Mansilla, Angelica Lisperger, Miguel Jarpa, Geraldine Larroquete, Fernando AvendañoAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparision between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV.
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Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences
Virology Journal, 2009Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Yingwei Wang, Valentina Gherardelli, Soledad Mansilla, Angelica Lisperger, Miguel Jarpa, Geraldine Larroquete, Marcos G Godoy, Fernando AvendañoAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon ( Salmo salar ); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae , together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus . The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparision between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV. Results RT-PCR for ISAV F and HE glycoprotein genes was performed directly on tissue samples collected from ISA-affected fish on different farms among 14 fish companies in Chile during the ISA outbreaks that started in June 2007. The genes of the F and HE glycoproteins were cloned and sequenced for 51 and 78 new isolates, respectively. An extensive comparative analysis of ISAV F and HE sequence data, including reference isolates sampled from Norway, Faroe Islands, Scotland, USA, and Canada was performed. Based on phylogenetic analysis of concatenated ISAV F and HE genes of 103 individual isolates, the isolates from the ISA outbreaks in Chile grouped in their own cluster of 7 distinct strains within Genotype I (European genotype) of ISAV, with the closest relatedness to Norwegian ISAVs isolated in 1997. The phylogenetic software program, BACKTRACK, estimated the Chile isolates diverged from Norway isolates about 1996 and, therefore, had been present in Chile for some time before the recent outbreaks. Analysis of the deduced F protein sequence showed 43 of 51 Chile isolates with an 11-amino acid insert between ^265N and ^266Q, with 100% sequence identity with Genotype I ISAV RNA segment 2. Twenty four different HE-HPRs, including HPR0, were detected, with HPR7b making up 79.7%. This is considered a manifestation of ISAV quasispecies HE protein sequence diversity. Conclusion Taken together, these findings suggest that the ISA outbreaks were caused by virus that was already present in Chile that mutated to new strains. This is the first comprehensive report tracing ISAV from Europe to South America.
Tomy Joseph - One of the best experts on this subject based on the ideXlab platform.
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Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus
Virology journal, 2007Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Biao Qian, Tomy JosephAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV.
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Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus
Virology Journal, 2007Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Biao Qian, Tomy JosephAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus , one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. Results In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the same antiserum revealed the protein(s) to be localized in the cytoplasm. Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge. Conclusion Collectively, our observations suggest that the product of ISAV segment 7 primary transcript (7-ORF1) is a structural protein. The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals. The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.
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Infectious salmon anemia virus: causative agent, pathogenesis and immunity.
Animal health research reviews, 2004Co-Authors: Frederick S. B. Kibenge, Molly J. T. Kibenge, Tomy Joseph, Khalid Munir, Emeka MonekeAbstract:Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90-140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.
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Antibody-mediated growth of infectious salmon anaemia virus in macrophage-like fish cell lines.
The Journal of general virology, 2003Co-Authors: Tomy Joseph, Molly J. T. Kibenge, Frederick S. B. KibengeAbstract:Infectious salmon anaemia virus (ISAV), a pathogen in marine aquaculture, belongs to the genus Isavirus, family Orthomyxoviridae. There is limited information on how ISAV interacts with host defences. To study ISAV-antibody interactions, virus neutralization (VN) assays were performed in the cell lines CHSE-214, SHK-1 and TO using three strains of ISAV and rabbit or fish anti-ISAV sera. Homologous VN titres of >1 : 1280 in CHSE-214 cells corresponded to titres of only 1 : 80 in the macrophage-like fish cell lines SHK-1 and TO, despite using 1000 and 2000 times less virus, respectively. However, rabbit antiserum to infectious pancreatic necrosis virus (IPNV) had a VN titre of 1 : 10,260 against IPNV in both CHSE-214 and TO cells. Poor ISAV neutralization in TO cells was attributed to Fc receptors mediating virus infectivity, because (1) neutralization by rabbit antiserum to ISAV was increased 48-fold in the presence of staphylococcal Protein A and (2) when using FITC-labelled virus and spectrofluorometry, a significant increase (P=0.018) in the intensity of fluorescence of intracellular virus was observed in assays of virus-antiserum mixtures in the absence of Protein A as compared to those in the presence of Protein A. Neutralization of ISAV with fish antisera was observed only in CHSE-214 cells, as Protein A could not restore neutralization in TO cells. These findings demonstrate for the first time antibody-mediated infection of macrophage-like fish cell lines by a fish virus, ISAV, and, as ISAV in Atlantic salmon targets leukocytic and endothelial cells, this may have implications for ISA pathogenesis and vaccination.
Samuel T Workenhe - One of the best experts on this subject based on the ideXlab platform.
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Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes
Virology Journal, 2008Co-Authors: Samuel T Workenhe, Molly J. T. Kibenge, Dorota W Wadowska, Glenda M Wright, David B. Groman, Frederick S. B. KibengeAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus , family Orthomyxoviridae . ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination. Results Haemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID_50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-α in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 – 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (
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Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes
Virology journal, 2008Co-Authors: Samuel T Workenhe, Molly J. T. Kibenge, Dorota W Wadowska, Glenda M Wright, David B. Groman, Frederick S. B. KibengeAbstract:Background Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.
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Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon
Virology Journal, 2007Co-Authors: Samuel T Workenhe, Molly J. T. Kibenge, Dorota W Wadowska, Glenda M Wright, Frederick S. B. KibengeAbstract:Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae . It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon ( Salmo salar ) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout ( Oncorhynchus mykiss ) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.
