scholarly journals Classical Swine Fever Virus ErnsDeletion Mutants: trans-Complementation and Potential Use as Nontransmissible, Modified, Live-Attenuated Marker Vaccines

2000 ◽  
Vol 74 (7) ◽  
pp. 2973-2980 ◽  
Author(s):  
M. N. Widjojoatmodjo ◽  
H. G. P. van Gennip ◽  
A. Bouma ◽  
P. A. van Rijn ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Classical Swine Fever Virus ◽  
Lethal Dose ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Structural Protein ◽  
Viral Particles

ABSTRACT An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein Erns of classical swine fever virus (CSFV) was used to rescue CSFV Erns deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of Erns from this cell line were indistinguishable from those of CSFV Erns. Two Erns deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of Erns (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of Erns antibodies, due to a deletion of 66 amino acids in Erns. The Erns deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by Erns-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that Erns from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) Erns in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.

scholarly journals Classical swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation by binding to NS5A

2012 ◽  
Vol 93 (5) ◽  
pp. 939-950 ◽  
Author(s):  
Chun Sheng ◽  
Jing Wang ◽  
Jing Xiao ◽  
Jun Xiao ◽  
Yan Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Inhibitory Effect ◽  
Fever Virus ◽  
Viral Translation ◽  
Ns5a Protein ◽  
Gdd Motif ◽  
Ns5b Protein

In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione S-transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390–414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A–NS5B interaction, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63–72, aa 637–653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A–NS5B interaction, for which aa 390–414 of NS5A and aa 63–72, aa 637–653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A–NS5B interaction in the virus life cycle.

scholarly journals Characterization of Essential Domains and Plasticity of the Classical Swine Fever Virus Core Protein

2010 ◽  
Vol 84 (21) ◽  
pp. 11523-11531 ◽  
Author(s):  
Christiane Riedel ◽  
Benjamin Lamp ◽  
Manuela Heimann ◽  
Till Rümenapf
Keyword(s):  
Amino Acids ◽  
Classical Swine Fever Virus ◽  
Core Protein ◽  
Yellow Fluorescent Protein ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Progeny Virus ◽  
Special Importance ◽  
The Core ◽  
Rna Interaction

ABSTRACT Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins Erns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala255 at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.

scholarly journals Antibody Responses of Pigs to Defined Erns Fragments after Infection with Classical Swine Fever Virus

2005 ◽  
Vol 12 (1) ◽  
pp. 180-186 ◽  
Author(s):  
Min Lin ◽  
Erin Trottier ◽  
John Pasick
Keyword(s):  
Amino Acids ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Characteristic Analysis ◽  
Western Blots ◽  
Consensus Region ◽  
Nickel Chelate

ABSTRACT Antibody responses of pigs to defined Erns fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA). Selection of various Erns fragments was based on an immunodominant Erns region encompassing three overlapping antigenic regions, amino acids 65 to 145 (Erns aa 65-145) (AR1), 84 to 160 (Erns aa 84-160) (AR2), and 109 to 220 (Erns aa 10 9-220) (AR3), identified earlier by our group (M. Lin, E. Trottier, J. Pasick, and M. Sabara, J. Biochem., in press). Defined Erns fragments, including AR1, AR2, AR3, Erns aa 65-160 (AR12), Erns aa 84-220 (AR23), Erns aa 65-220 (AR123), Erns aa 109-145 (the consensus region defined by the three overlapping regions), and Erns aa 109-160 (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV. Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the Erns fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100%. Detection of antibody responses to refolded Erns aa 109-145 and Erns aa 109-160 by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational. When cutoff values were raised to give a specificity of 100%, four Erns fragments (AR2, AR23, Erns aa 109-145, and Erns aa 109-160) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%). Erns aa 109-145 and Erns aa 109-160 were capable of detecting antibody responses in infected pigs as early as 7 days postinfection. Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection. An advantage of such a test would be its utilization for serological survey in a classical swine fever-free country (e.g., Canada) in biocontainment level 2 laboratories.

scholarly journals Cloning and identification of PK15 cells for enhanced replication of classical swine fever virus

2020 ◽  
Vol 64 (1) ◽  
pp. 9-14
Author(s):  
Mei Yin ◽  
Dongfang Hu ◽  
Peng Li ◽  
Lingyun Kong ◽  
Hongmei Ning ◽  
...  
Keyword(s):  
Cell Line ◽  
Classical Swine Fever Virus ◽  
Vaccine Development ◽  
Cell Clone ◽  
Classical Swine Fever ◽  
High Titre ◽  
Fever Virus ◽  
Economic Losses ◽  
Cell Clones

AbstractIntroductionClassical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection.Material and MethodsTo achieve a homogeneous PK15 cell line which enabled high titre replication of CSFV, we used the limiting dilution cell cloning method.ResultsWe developed two cell clones, PK15-1A6 and PK15-3B1, which respectively have high- and low-permissive phenotypes to CSFV infection. The PK15-1A6, PK15-3B1, and PK15 parent cells showed different characteristics in cell proliferation rate, susceptibility to CSFV infection, and CSFV production. The mean virus titres per millilitre reflected by TCID50 values in PK15-1A6, PK15-3B1, and PK15 parent cells were 106.85, 103.63, and 104.74, respectively.ConclusionThe PK15-1A6 cell clone is more permissive to CSFV infection than the PK15 parent cells. The screened high-permissive cells will be useful for CSFV propagation and vaccine development in vitro, and facilitate research on the pathogenicity of CSFV.

scholarly journals In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line

2004 ◽  
Vol 35 (6) ◽  
pp. 625-633 ◽  
Author(s):  
Emilia Campos ◽  
Concepci�n Revilla ◽  
Sonia Chamorro ◽  
Bel�n Alvarez ◽  
Angel Ezquerra ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Line ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Porcine Aortic Endothelial Cell ◽  
Aortic Endothelial Cell ◽  
Endothelial Cell Line ◽  
Vitro Effect

scholarly journals Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus

2002 ◽  
Vol 83 (3) ◽  
pp. 551-560 ◽  
Author(s):  
Elisenda Armengol ◽  
Karl-Heinz Wiesmüller ◽  
Daniel Wienhold ◽  
Mathias Büttner ◽  
Eberhard Pfaff ◽  
...  
Keyword(s):  
T Cell ◽  
Mhc Class Ii ◽  
Classical Swine Fever Virus ◽  
Cell Epitope ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Target Cells ◽  
T Cell Epitopes ◽  
Structural Protein

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446–1460 of the CSFV non-structural protein NS2–3 could induce interferon-γ secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

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