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

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.

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

Journal of Virology ◽

10.1128/jvi.00551-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8602-8613 ◽

Cited By ~ 41

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.

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Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

Virology ◽

10.1016/j.virol.2014.03.005 ◽

2014 ◽

Vol 456-457 ◽

pp. 121-130 ◽

Cited By ~ 15

Author(s):

I.J. Fernández-Sainz ◽

E. Largo ◽

D.P. Gladue ◽

P. Fletcher ◽

V. O’Donnell ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fusion Peptide ◽

Fever Virus ◽

Amino Acid Substitutions ◽

Specific Amino Acid ◽

Glycoprotein E2

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Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

Journal of Virology ◽

10.1128/jvi.78.16.8812-8823.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8812-8823 ◽

Cited By ~ 48

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.

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Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication

Viruses ◽

10.3390/v12040388 ◽

2020 ◽

Vol 12 (4) ◽

pp. 388 ◽

Cited By ~ 1

Author(s):

Elizabeth A. Vuono ◽

Elizabeth Ramirez-Medina ◽

Keith Berggren ◽

Ayushi Rai ◽

Sarah Pruitt ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Host Protein ◽

Yeast Two Hybrid ◽

Host Proteins ◽

E2 Protein ◽

Two Hybrid

Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2–CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2–CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.

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ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109034 ◽

2021 ◽

Vol 255 ◽

pp. 109034

Author(s):

Liang Zhang ◽

Mingxing Jin ◽

Mengzhao Song ◽

Shanchuan Liu ◽

Tao Wang ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Ns5a Protein

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Genome Variability of Classical Swine Fever Virus during the 2018–2020 Epidemic in Japan

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109128 ◽

2021 ◽

pp. 109128

Author(s):

Tatsuya Nishi ◽

Katsuhiko Fukai ◽

Tomoko Kato ◽

Kotaro Sawai ◽

Takehisa Yamamoto

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Genome Variability

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A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

Vaccines ◽

10.3390/vaccines9050418 ◽

2021 ◽

Vol 9 (5) ◽

pp. 418

Author(s):

Youngmin Park ◽

Yeonsu Oh ◽

Miaomiao Wang ◽

Llilianne Ganges ◽

José Alejandro Bohórquez ◽

...

Keyword(s):

Vertical Transmission ◽

Classical Swine Fever Virus ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Dose ◽

Classical Swine Fever ◽

Fever Virus ◽

Tissue Samples ◽

Marker Vaccine ◽

E2 Glycoprotein

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

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