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B L Nicholson

Publications and source records attributed to B L Nicholson.

At least 19 recordsLinked to original sources
PubMed2004

Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain.

Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.

Animals↗
PubMed2003

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA.

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.

Amino Acid Sequence↗
PubMed2001

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.

Amino Acid Sequence↗
PubMed1999

Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada.

Infectious salmon anemia virus (ISAV) was isolated at a marine grow-out site in New Brunswick, Canada, from Atlantic salmon Salmo salar which experienced mortalities due to hemorrhagic kidney syndrome (HKS). Of 20 fish sampled in this study, 14 showed histologically various degrees of interstitial hemorrhaging, tubular epithelial degeneration and necrosis, and tubular casts in the posterior kidney, typical of HKS. Posterior kidney and spleen homogenates produced a cytopathic effect on chinook salmon embryo (CHSE-214) cells 10 to 14 d after inoculation. Pleomorphic virus particles in the size range 80 to 120 nm were seen by electron microscopy. The virus was confirmed as ISAV using reverse transcriptase-polymerase chain reaction (RT-PCR). This is a systematic diagnostic study of the isolation of ISAV on the North American continent and the first description of the growth of ISAV on the CHSE-214 cell line.

Anemia↗
PubMed1999

Genomic relationships of the North American isolate of infectious salmon anemia virus (ISAV) to the Norwegian strain of ISAV.

Nucleotide and deduced amino acid sequences were determined for a 436 bp reverse transcriptase-polymerase chain reaction (RT-PCR) cDNA fragment from genome segment 8 and a 1151 bp RT-PCR cDNA fragment from genome segment 2 of the North American isolate of infectious salmon anemia virus (ISAV) and compared to the published sequences of Norwegian isolates of ISAV. The North American ISAV isolate exhibited 82.9% identity with the Sotra 92/93 ISAV isolate from Norway in the partial cDNA sequence of genome segment 2, which encodes a polymerase component protein (PB1). The North American ISAV exhibited 88 and 89% identity with 2 partial cDNA sequences of genome segment 8 (nonstructural, NS, gene) reported for the Glesvaer/2/90 isolate from Norway. The North American ISAV exhibited 96.6% similarity with the Sotra 92/93 ISAV isolate from Norway in the deduced amino acid sequences of the PB1 protein. The deduced amino acid sequence of the protein encoded in the partial cDNA fragment of open reading frame (ORF) 1 of genome segment 8 of the North American ISAV exhibited only 71.2 and 66.7% similarity with the 2 sequences of the Norwegian Glesvaer/2/90 isolate. However, the North American ISAV isolate exhibited 96.2 and 87.2% similarity with the 2 sequences of the Norwegian Glesvaer/2/90 isolate in the deduced amino acid sequences of the protein encoded in the partial cDNA of ORF 2. Comparison of these partial cDNA nucleotide and deduced amino acid sequences confirmed that the North American isolate is ISAV. However, the differences observed in these genomic sequences suggest that the North American isolate may represent a distinct genomic variant from the previously described Norwegian strains.

Amino Acid Sequence↗
PubMed1999

Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses.

A multiplex reverse transcriptase (RT)-PCR assay was developed for the simultaneous detection of three different fish viruses: infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV). The sensitivity levels of the multiplex RT-PCR assay were 100, 1, and 32 50% tissue culture infective doses/ml for IPNV, IHNV, and VHSV, respectively.

Animals↗
PubMed1996

Genomic variation of aquatic birnaviruses analyzed with restriction fragment length polymorphisms.

Aquatic birnaviruses are the most ubiquitous and diverse group of viruses in the family Birnaviridae. Several cause different diseases in a variety of fish species, such as infectious pancreatic necrosis virus in salmonids in North America, Europe, and Asia and European eel virus in eel in Asia. Most isolates are antigenically related and belong to a single serogroup (serogroup A) comprising nine serotypes. Previous studies with monoclonal antibodies have demonstrated considerable variation in epitope profiles even among strains within a single serotype. The few studies of genomic variation among these viruses, which have focused on the NS/VP3 coding region, demonstrated the existence of several genogroups that generally did not correlate with antigenic groups. In this study, PCR was used to amplify a 1,180-bp cDNA genomic fragment representing most of the VP2 (the major outer capsid protein) coding region from five serotype A type strains and 17 Asian isolates. The PCR products were digested with nine different restriction enzymes. Restriction fragment length polymorphism profiles demonstrated heterogeneity among the tested viruses; however, the isolates from Asia were closely related to each other. Cluster analysis of the restriction fragment length polymorphism patterns demonstrated that these viruses could be divided into four major genogroups. In contrast to previous studies of variation in the NS/VP3 coding region, these genogroups based on variation in the VP2 coding region correlated with a serological classification based on VP2-specific monoclonal antibody reaction patterns. Furthermore, all Asian isolates tested belonged to one genogroup typified by the serotype type strain Ab.

Animals↗
PubMed1995

Comparison of different procedures for serotyping aquatic birnavirus.

The current classification of aquatic birnaviruses is based on seroneutralization assays with polyclonal antibodies. In this study a comparison of several procedures used for serotyping aquatic birnaviruses was made with 10 virus strains (4 reference strains from salmonids and 6 birnaviruses isolated from turbot [Scophthalmus maximus]). The relationships among the birnavirus strains were studied by seroneutralization assay with polyclonal antibodies and by immunodot assay with both polyclonal and monoclonal antibodies. The results were compared with a presumptive classification obtained from analysis of restriction enzyme patterns of cDNA products obtained by PCR amplification. No correlation was found among the results obtained by the different procedures. The seroneutralization and the immunodot assays with polyclonal antibodies were not useful in classifying these birnaviruses strains; however, patterns of reaction with monoclonal antibodies emphasized the individuality of the strains, particularly in the case of two strains (231 and 460) whose patterns did not correspond to established serotypes. The application of PCR and restriction enzyme analysis is a promising system for approaching the classification of this viral group on the basis of genomic differences and similarities. The variable results obtained in this comparison lead us to think that the current classification of aquatic birnavirus may not be the most accurate and there is a need for modification incorporating recent isolates, not only from salmonid species but also from marine fish.

Animals↗
PubMed1995

Detection and identification of aquatic birnaviruses by PCR assay.

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

Animals↗
PubMed1995

Application of 3 techniques for diagnosing birnavirus infection in turbot.

Three procedures for avoiding viral amplification by cell culture were evaluated for the diagnosis of birnavirus infections of turbot (Scophthalmus maximus) tissues. Immunodot assay using monoclonal and polyclonal antibodies was not satisfactory as a detection system because of false positive reactions. Although immunofluorescent assay of liver and kidney smears was an adequate method for a rapid diagnosis of the infection, blood smears from infected fish did not show higher levels of fluorescence than those from uninfected fish. However, the detection of birnavirus by polymerase chain reaction (PCR) amplification directly in fish tissues was successful and seems to be a promising system for the diagnosis of fish birnavirus.

Animals↗
PubMed1989

Use of a group-reactive and other monoclonal antibodies in an enzyme immunodot assay for identification and presumptive serotyping of aquatic birnaviruses.

A panel of monoclonal antibodies (MAbs) was prepared and used to develop an enzyme immunodot assay for the rapid identification and presumptive serotyping of aquatic birnaviruses. Comparison of the reaction patterns of these MAbs with representative virus isolates indicated that one MAb recognizes a serogroup-reactive epitope and can therefore be used for identification of all serogroup A aquatic birnaviruses, the predominant serotype worldwide. Other MAbs exhibited more restrictive specificities, permitting the presumptive serotyping of viruses of the three recognized serotypes and the identification of some individual strains. This assay, in which MAbs are used, is more efficient in terms of time, cost, and case of performance and provides significant advantages in specificity and standardization compared with currently used tests.

Animals↗
PubMed1986

Monoclonal antibodies to infectious pancreatic necrosis virus: analysis of viral epitopes and comparison of different isolates.

A panel of five monoclonal antibodies (MAbs) produced against the West Buxton isolate of infectious pancreatic necrosis virus was used to investigate viral antigens and to compare different aquatic birnavirus isolates antigenically. Reciprocal blocking ELISA and neutralization assays indicated that these MAbs identified four, and possibly five, structurally and/or functionally different epitopes on the virion. Western immunoblot analysis demonstrated that one epitope was present on VP2, the large (51,000 mol. wt., 51K) capsid protein, and another epitope was located on the two smallest structural proteins, VP3 (32K) and VP4 (30K). Three MAbs did not react with any of the solubilized viral proteins; the epitopes recognized by these MAbs may have been altered when the virion was solubilized with SDS. Comparison of reactivity patterns of the five MAbs with various aquatic birnaviruses in ELISA and neutralization tests demonstrated that 14 isolates tested from four serotypes represented a minimum of nine antigenically distinct viruses; i.e., distinct patterns of reactivity were shown among several viruses within the same serotype. Two MAbs identified different epitopes that were highly conserved among, and largely restricted to, members of the West Buxton (U.S.A.) serotype, whereas two other MAbs recognized an epitope(s) present only on some members of this serotype. The other MAb defined an epitope that was more widely distributed among the aquatic birnaviruses and found on all representatives tested from two serotypes.

Antibodies, Monoclonal↗
PubMed1982

Enzyme-linked immunosorbent assay for identification of infectious pancreatic necrosis virus.

The sensitivity, reproducibility, and specificity of an enzyme-linked immunosorbent assay for the identification of infectious pancreatic necrosis virus was determined. It was shown that 10(5.5) tissue culture infectious doses could be assayed by this method. The assay provided significant advantages in terms of time and ease of performance over the routinely employed serological tests. When an antiserum against a single isolate was used, it was possible to identify nine of ten North American isolates, four of six European isolates, and one isolate from Taiwan.

Animals↗
PubMed1980

Growth of fish cell lines on microcarriers.

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 x 10 to 2.9 x 10 cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.

Journal Article↗
PubMed1978

Rapid identification of infectious pancreatic necrosis virus in infected cell cultures by immunoperoxidase techniques.

Direct and indirect immunoperoxidase (IP) techniques were evaluated for their potential in identifying infectious pancreatic necrosis (IPN) virus. Both techniques were shown to offer a relatively simple, rapid and efficient means for the specific identification of IPN virus in infected cells. The direct IP method resulted in less nonspecific staining; however, the indirect method was clearly specific and utilized commercially available reagents.

Cells, Cultured↗
PubMed1974

Homologous viral interference in trout and Atlantic salmon cell cultures infected with infectious pancreatic necrosis virus.

The occurrence of homologous interference in the replication of infectious pancreatic necrosis virus was demonstrated after successive passages of partially purified virus at high input multiplicities in trout and Atlantic salmon cell cultures. Pretreatment of cell cultures with interfering virus inhibited the replication of homologous standard infectious virus but not unrelated viruses. The ability of infectious pancreatic interfering virus to interfere with homologous virus was abolished with UV irradiation, immune serum, and freeze-thawing.

Animals↗