Development of a ssRNA internal control reagent for an infectious bursal disease virus reverse transcription/polymerase chain reaction-restriction fragment length polymorphism diagnostic assay.
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Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences. A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results. (+info)
CpG DNA induces protective antiviral immune responses in Atlantic salmon (Salmo salar L.).
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Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN show promise as vaccine adjuvants and immunoprotective agents in animal models. Here we report that pretreatment with CpG ODN in animals induces nonspecific protection against viral infection. A panel of different synthetic CpG ODN was tested for the in vitro effects in Atlantic salmon (Salmo salar L.) leukocytes. The ODN were tested for their capacity to stimulate proliferation of peripheral blood leukocytes and to induce production of interferon-like factors in head kidney leukocytes. These studies revealed that the sequence and number of the CpG motifs as well as the lengths of the ODN contribute to their stimulatory activity. ODN with the 6-mer CpG motif (5'-GTCGTT-3') showed the highest stimulatory activity and were shown to induce protection against infectious pancreatic necrosis virus when injected in Atlantic salmon. Expression of the Mx transcript, as an indicator of alpha/beta interferon induction, was induced in the CpG-injected fish. These results suggest that CpG DNA in fish induces early, nonspecific antiviral protection. (+info)
Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines.
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The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci. (+info)
Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA.
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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. (+info)
Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain.
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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. (+info)
Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus.
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Infectious pancreatic necrosis viruses (IPNVs) belonging to the family Birnaviridae display a high degree of antigenic variability, pathogenicity, and differences in outbreak mortality in salmonid species. To determine if virus isolates of Sp serotype differ in virulence, fry of Atlantic salmon (Salmo salar L.) were challenged with nine different field strains. These viruses caused either high mortality and severe pathological changes or low mortality and no lesions. To study the molecular basis for the variation in virulence of IPNV, complete nucleotide sequences of segment A of all these strains as well as segment B of three selected strains were determined. All viruses tested had a unique genome sequence. Only minor differences were noted in the genes encoding VP1, VP3, and VP4 proteins, whereas most changes were observed in the gene encoding the VP2 protein. A high level of variation was found in the small open reading frame (ORF), which encodes a 15-kDa nonstructural (NS) polypeptide also known as VP5. One of the strains lacked the initiation codon for this protein, whereas the other four could encode a truncated version of the NS protein. Additional data obtained by sequencing of the NS and VP2 genes directly from diseased fish demonstrated changes in the VP2 gene after two passages in cell culture, which could possibly be associated with attenuation. Comparison of the deduced amino acid sequences of the NS and VP2 genes reveals that the virulent strains possess a 12-kDa coding NS gene and have residues Thr, Ala, Thr/Ala, and Tyr/His at positions 217, 221, 247, and 500 of the VP2 gene, respectively-the motifs identified in this study to be involved in the virulence of IPNV. (+info)
A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV.
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Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3'-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens. (+info)
Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination.
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Although vaccines against infectious pancreatic necrosis (IPN) based on inactivated virus or recombinant structural viral proteins are commercially available, the protection is not complete and the disease is still a problem for the Atlantic salmon Salmo salar farming industry. In the present study, 5 different plasmids that expressed whole or parts of the large open reading frames (ORF) of Segment A of the IPN virus (IPNV) were constructed. The plasmids were shown to express proteins in cell cultures and in zebrafish Danio rerio in vivo. The specificities of the expressed proteins were confirmed by staining with IPNV-specific monoclonal antibodies (MAb) The plasmids were then used alone or in different combinations to vaccinate groups of Atlantic salmon, which subsequently were challenged in an experimental assay for IPN. A high level of protection was induced only by the plasmid combination that contained a plasmid expressing all the large ORF polyprotein. (+info)