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The specific detection of infectious pancreatic necrosis virus in infected cells and fish by the immuno dot blot method (1989)

HSU, YA-LI ; CHIANG, SHU-YUAN ; LIN, SHOU-TZU ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Journal of fish diseases 12 (1989), S. 0

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ISSN: 1365-2761

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Abstract. The immuno dot blot technique is a rapid, sensitive and quantitative method for the diagnosis of infectious pancreatic necrosis virus (ipnv) in both infected tissue culture lysates and tissue homogenates of fish. Uninfected tissue culture lysates and fish homogenates do not react in the peroxidase dot blot system after appropriate treatment. The immuno dot blot method takes 4h to complete, and the dot blot membrane can be stored in the dark for a long period. The viral proteins in the infected tissue culture cells could be detected at 3h post-infection if an initial high MOI was used. The minimum dose of ipnv-ab strain which could be detected using homologous serum was 2·5 ng purified ipnv protein/dot. The smallest infectious dose of VR-299, SP, AB, EVE and CY-1 ipnv which could be detected by the immuno dot blot system was 104 to 105 TCID50. When one μg of purified ipnv was used, a 1·6 × 104 dilution of rabbit anti-AB antisera could be detected.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2761.1989.tb00565.x

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Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators (2000)

Chen, Jyh-Yih ; Chen, Jyh-Yeu ; Chu, Wan-Ken ; [et al.]

Springer

Marine biotechnology 2 (2000), S. 364-375

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ISSN: 1436-2236

Keywords: Key words: Infectious pancreatic necrosis virus (IPNV), ribozyme, oligonucleotide facilitator, RNA cleavage, catalysis.

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract: Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a bisegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we cloned and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 bp) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp in length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA of IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates containing a T7 RNA polymerase promoter, and were used to characterize several properties of the cleavage reaction at 25°C in 12 mM Mg2+. Under this condition (25°C, 12 mM Mg2+), the hammerhead ribozymes formed an estimated fraction of product during the reaction of only 30% in cleaving long RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3′ or 5′ end of the ribozyme enhanced the rate of cleavage of the long RNA substrates containing 569 and 969 nucleotides, respectively, in trans. The hammerhead ribozymes with 3′-end facilitators reacted more efficiently (i.e., 65%).