scholarly journals Quantitative Flow Cytometry to Measure Viral Production Using Infectious Pancreatic Necrosis Virus as a Model: A Preliminary Study

2018 ◽  
Vol 8 (10) ◽  
pp. 1734 ◽  
Author(s):  
Diego Vázquez ◽  
Carmen López-Vázquez ◽  
José Olveira ◽  
Isabel Bandín ◽  
Carlos Dopazo
Keyword(s):  
Flow Cytometry ◽  
Viral Replication ◽  
Pancreatic Necrosis ◽  
Polynomial Regression ◽  
Infectious Pancreatic Necrosis Virus ◽  
Necrosis Virus ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus ◽  
Vitro Analysis

In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Finally, the results support the use of this technology, not only for virus quantification, but also to study viral replication from a quantitative approach.

scholarly journals Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon

2001 ◽  
Vol 82 (4) ◽  
pp. 805-812 ◽  
Author(s):  
Siegfried Weber ◽  
Dieter Fichtner ◽  
Thomas C. Mettenleiter ◽  
Egbert Mundt
Keyword(s):  
Pancreatic Necrosis ◽  
Nonstructural Protein ◽  
Infectious Pancreatic Necrosis Virus ◽  
Start Codon ◽  
Necrosis Virus ◽  
Virus Growth ◽  
Initiation Of Translation ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae with two double-stranded RNA genome segments, encodes five proteins designated VP1 to VP5. To study the function of the 17 kDa nonstructural protein VP5 during virus replication several mutated IPNV genome segments A were constructed and included in a reverse genetics system for IPNV to obtain recombinant virus. Mutations between nt 68 and 85 or nt 94 and 103 in the noncoding region failed to yield viable virus. Only mutations located between nt 86 and 92 and downstream of nt 104 were tolerated, and viable virus could be generated. All IPNV generated showed no difference in replication compared with the wild-type IPNV, indicating that the absence of expression of VP5 did not influence virus growth in vitro. Furthermore, the results presented here indicate that initiation of translation of VP5 occurs at position 113, the second in-frame start codon.

scholarly journals The Infectious Pancreatic Necrosis Virus (IPNV) and its Virulence Determinants: What is Known and What Should be Known

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 94 ◽  
Author(s):  
Carlos P. Dopazo
Keyword(s):  
Pancreatic Necrosis ◽  
Infectious Pancreatic Necrosis Virus ◽  
General Term ◽  
Virulence Determinants ◽  
Necrosis Virus ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus ◽  
Viral Components

Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among salmonid farmers, since losses in salmonid fish—mostly very young rainbow trout (Salmo gairdnery) fry and Atlantic salmon (Salmo salar) post-smolt—frequently reach 80–90% of stocks. The virus causing the typical signs of the IPN disease in salmonids, named infectious pancreatic necrosis virus (IPNV), has also been isolated from other fish species either suffering related diseases (then named IPNV-like virus) or asymptomatic; the general term aquabirnavirus is used to encompass all these viruses. Aquabirnaviruses are non-enveloped, icosahedral bisegmented dsRNA viruses, whose genome codifies five viral proteins, three of which are structural, and one of them is an RNA-dependent RNA polymerase. Due to the great importance of the disease, there have been great efforts to find a way to predict the level of virulence of IPNV isolates. The viral genome and proteins have been the main focus of research. However, to date such a reliable magic marker has not been discovered. This review describes the processes followed for decades in the attempts to discover the viral determinants of virulence, and to help the reader understand how viral components can be involved in virulence modulation in vitro and in vivo. There is also a brief description of the disease, of host defenses, and of the molecular structure and function of the virus and its viral components.

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