scholarly journals Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

2012 ◽  
Vol 86 (16) ◽  
pp. 8602-8613 ◽  
Author(s):  
Tomokazu Tamura ◽  
Yoshihiro Sakoda ◽  
Fumi Yoshino ◽  
Takushi Nomura ◽  
Naoki Yamamoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Amino Acid Substitutions ◽  
Virulence Determinants ◽  
Live Attenuated Vaccine

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE−was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE−vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE−/P-11 virus isolated from the tonsils after the 11th passagein vivohad acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studiesin vitroindicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiencyin vitroand pathogenicity in pigs.

scholarly journals Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

2004 ◽  
Vol 78 (16) ◽  
pp. 8812-8823 ◽  
Author(s):  
H. G. P. van Gennip ◽  
A. C. Vlot ◽  
M. M. Hulst ◽  
A. J. de Smit ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Reverse Genetics ◽  
Animal Experiments ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Target Cells ◽  
Adaptive Mutations

ABSTRACT Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476S variant into a His residue did not affect virulence. However, the 710His in the C1.1.1/CoBrB virus, together with adaptive mutations 276R, 476R, and 477I in Erns, resulted in reduced virulence in pigs. These results indicated that mutations in Erns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 Erns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

scholarly journals Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo

2006 ◽  
Vol 37 (1) ◽  
pp. 155-168 ◽  
Author(s):  
Henriette Rau ◽  
Hilde Revets ◽  
Carole Balmelli ◽  
Kenneth C. McCullough ◽  
Artur Summerfield
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Immunological Properties ◽  
Ns3 Protein

Classical swine fever virus marker vaccine strain CP7_E2alf: genetic stability in vitro and in vivo

2015 ◽  
Vol 160 (12) ◽  
pp. 3121-3125 ◽  
Author(s):  
Katja V. Goller ◽  
Carolin Dräger ◽  
Dirk Höper ◽  
Martin Beer ◽  
Sandra Blome
Keyword(s):  
Vaccine Strain ◽  
Classical Swine Fever Virus ◽  
Genetic Stability ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Marker Vaccine

scholarly journals Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine

2016 ◽  
Vol 90 (22) ◽  
pp. 10299-10308 ◽  
Author(s):  
L. G. Holinka ◽  
E. Largo ◽  
D. P. Gladue ◽  
V. O'Donnell ◽  
G. R. Risatti ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structural Analysis ◽  
Cell Cultures ◽  
Classical Swine Fever Virus ◽  
Synthetic Peptides ◽  
Classical Swine Fever ◽  
Fusion Peptide ◽  
Fever Virus ◽  
Amino Acid Substitutions ◽  
Artificial Membranes

ABSTRACTE2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an872GG873dipeptide, as well as two aromatic residues (871W and875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICvin vitroand virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2.IMPORTANCEThis report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuatedin vivoefficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

scholarly journals Novel poly-uridine insertion in the 3′UTR and E2 amino acid substitutions in a low virulent classical swine fever virus

2017 ◽  
Vol 201 ◽  
pp. 103-112 ◽  
Author(s):  
Liani Coronado ◽  
Matthias Liniger ◽  
Sara Muñoz-González ◽  
Alexander Postel ◽  
Lester Josue Pérez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Amino Acid Substitutions

scholarly journals Genetically modified pigs are protected from classical swine fever virus

2018 ◽  
Author(s):  
Zicong Xie ◽  
Daxin Pang ◽  
Hongming Yuan ◽  
Huping Jiao ◽  
Chao Lu ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Well Being ◽  
Fever Virus ◽  
Economic Losses ◽  
Transgenic Pigs ◽  
F1 Generation

AbstractClassical swine fever (CSF) caused by classical swine fever virus (CSFV) is among the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities try to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine ROSA26 (pROSA26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Importantly, in vitro and in vivo viral challenge assays demonstrated that these TG pigs could effectively limit the growth of CSFV and reduced CSFV-associated clinical signs and mortality, and the disease resistance was stably transmitted to F1-generation. The use of these TG pigs can improve the well-being of livestock and substantially reduce virus-related economic losses. Additionally, this antiviral approach may provide a reference for future antiviral research.Author summaryClassical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease with significant economic losses. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread in some regions and seems to be waiting for the reintroduction and the next round of disease outbreaks. These highlight the necessity and urgency of developing more effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV transgenic pigs and confirmed that these transgenic pigs could effectively limit the growth of CSFV in vivo and in vitro and that the disease resistance traits in the TG founders can be stably transmitted to their F1-generation offspring. This study suggests that these TG pigs can improve the well-being of livestock and contribute to offer potential benefits over commercial vaccination. The use of these TG pigs can improve the well-being of livestock and substantially reduce CSFV-related economic losses.

Classical swine fever virus is genetically stable in vitro and in vivo

1999 ◽  
Vol 144 (9) ◽  
pp. 1669-1677 ◽  
Author(s):  
H. Vanderhallen ◽  
C. Mittelholzer ◽  
M. A. Hofmann ◽  
F. Koenen
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus
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