scholarly journals Development and Application of a RT-NestPCR Assay for Differential Detection of C-Strain and Wild-Type Viruses of Classical Swine Fever Virus

2013 ◽  
Vol 2 (3) ◽  
pp. 27
Author(s):  
Haiguang Wang ◽  
Yibao Ning ◽  
Cong Ying ◽  
Can Liu ◽  
Ruirui Wei ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Gel Electrophoresis ◽  
Classical Swine Fever ◽  
High Sensitivity ◽  
Fever Virus ◽  
Test Method ◽  
Wild Type Virus ◽  
Diagnostic Tools ◽  
Differential Detection ◽  
Wild Type

<p>Due to the urgent need of differentiation of infected from vaccinated animals in control and eradication of classical swine fever (CSF) and the shortcomings of current differential diagnostic tools, this study is aiming to establish a RT-nestPCR assay for differential detection of wild-type viruses and lapinized Chinese vaccine strain (C-strain) of classical swine fever virus (CSFV) of high sensitivity. Two pairs of CSFV-specific primers were designed in the conservative regions of NS5B (a non-structural protein encoded by the CSFV genome, which performs the RNA dependent RNA polymerase activity) and 3? un-translated regions (3?-UTR) to encompass the T-rich insertion uniquely existing in the 3?-UTR of C-strain genome. Thus the amplification fragment of C-strain is longer than that of the wild-type viruses for it contains the T-rich insertion region. Two pairs of primers were used in combination and the wild-type viruses and C-strain of CSFV could be detected and accurately distinguished with a high sensitivity through super fine resolution (SFR) argarose gel electrophoresis that displays the different lengths of the amplicons. The detection limit of the C-strain and Shimen strain were respectively 4.5×10<sup>-2 </sup>pg and 3.2×10<sup>-2 </sup>pg of viral RNA. The results of the specificity test showed that this method can detect different strains of CSFV without amplifying other non-CSFV pathogens. The results of the detection of 400 clinical samples indicated that 16 samples were CSFV positive in total; in which 4 samples were C-Strain positive and 12 were wild-type CSFV positive. The total CSFV positive rate was 4%. The detection results of the 14 batches of C-Strain vaccines showed that all samples displayed bands of C-Strain amplicons in the SFR argarose gel electrophoresis and all vaccines were free of wild-type virus contamination. In conclusion, the RT-nestPCR assay established in the present study could supply a sensitive and specific test method for distinguishing wild-type CSFV infected animals from those vaccinated with C-strain vaccines in the field.</p>

scholarly journals Differential detection of classical swine fever virus challenge strains in C-strain vaccinated pigs

2014 ◽  
Vol 10 (1) ◽  
Author(s):  
Helen E Everett ◽  
Bentley S Crudgington ◽  
Olubukola Sosan-Soulé ◽  
Helen R Crooke
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Differential Detection ◽  
Virus Challenge

scholarly journals Mutation of Cysteine 171 of Pestivirus Erns RNase Prevents Homodimer Formation and Leads to Attenuation of Classical Swine Fever Virus

2009 ◽  
Vol 83 (10) ◽  
pp. 4823-4834 ◽  
Author(s):  
Birke Andrea Tews ◽  
Eva-Maria Schürmann ◽  
Gregor Meyers
Keyword(s):  
Classical Swine Fever Virus ◽  
Gel Electrophoresis ◽  
Bond Formation ◽  
Classical Swine Fever ◽  
Neutralizing Antibody ◽  
Fever Virus ◽  
Farm Animals ◽  
Protein Gel ◽  
Intermolecular Disulfide Bond ◽  
Infected Cells

ABSTRACT Pestiviruses represent important pathogens of farm animals that have evolved unique strategies and functions to stay within their host populations. Erns, a structural glycoprotein of pestiviruses, exhibits RNase activity and represents a virulence factor of the viruses. Erns forms disulfide linked homodimers that are found in virions and virus-infected cells. Mutation or deletion of cysteine 171, the residue engaged in intermolecular disulfide bond formation, results in loss of dimerization as tested in coprecipitation and native protein gel electrophoresis analyses. Nevertheless, stable virus mutants with changes affecting cysteine codon 171 could be recovered in tissue culture. These mutants grew almost as well as the parental viruses and exhibited an RNase-positive phenotype. Erns dimerization-negative mutants of classical swine fever virus were found to be attenuated in pigs even though the virus clearly replicated and induced a significant neutralizing antibody response in the animals.

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.

Purification of Classical Swine Fever Virus E2 Subunit Vaccines basing on High Affinity Peptide Ligand.

2020 ◽  
Vol 27 ◽  
Author(s):  
Fangyu Wang ◽  
Qiuying Yu ◽  
Man Hu ◽  
Guangxu Xing ◽  
Dong Zhao ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Rapid Development ◽  
Classical Swine Fever ◽  
High Sensitivity ◽  
Fever Virus ◽  
Affinity Constant ◽  
Peptide Ligand ◽  
Binding Interaction ◽  
Sensitivity And Selectivity

Background: The purification of expressed proteins is the most critical part of subunit-vaccine production. Protein-purification methods such as affinity chromatography and ion exchange still have the shortcomings of being time consuming and complicated. With the rapid development of computational molecular-simulation technology, structure-based peptide-ligand design has become feasible. Objection: We aimed to apply molecular docking for a peptide ligand designed for classical swine fever virus (CSFV) E2 purification. Methods: Computational-derived peptides were synthesized, and the in vitro binding interaction with E2 was investigated. The effects of purification on E2 were also evaluated. Results: The best peptide recognizing E2 was P6, which had a sequence of KKFYWRYWEH. Based on kinetic surface plasmon resonance (SPR) analysis, the apparent affinity constant of P6 was found to be 148 nM. Importantly, P6 showed suitable binding affinity and specificity for E2 purification from transgenic rice seeds. Evaluation of immune antibodies in mice showed that the antibody-blocking rate on day 42 after inoculation reached 86.18% and 90.68%. Conclusion: The computational-designed peptide in this study has high sensitivity and selectivity and is thus useful for the purification of CSFV E2. The novel method of design provided a broad platform and powerful tool for protein-peptide screening, as well as new insights into CSFV vaccine design.

scholarly journals A Polyuridine Insertion in the 3′ Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Miaomiao Wang ◽  
Matthias Liniger ◽  
Sara Muñoz-González ◽  
José Alejandro Bohórquez ◽  
Yoandry Hinojosa ◽  
...  
Keyword(s):  
Immune Responses ◽  
Classical Swine Fever Virus ◽  
Untranslated Region ◽  
Severe Disease ◽  
Classical Swine Fever ◽  
Viral Disease ◽  
Fever Virus ◽  
Diagnostic Tools ◽  
Virulence Determinants ◽  
Viral Virulence

ABSTRACT Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3′ untranslated region (3′ UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3′ UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3′ UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission. IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3′ untranslated region (3′ UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3′ UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

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