J Vet Sci.  2012 Mar;13(1):81-91. 10.4142/jvs.2012.13.1.81.

Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain

• Park GS
• Lim SI
• Hong SH
• Song JY

• KMID: 1365007
• DOI: http://doi.org/10.4142/jvs.2012.13.1.81

Abstract

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.

Keyword

classical swine fever virus; Flc-LOM; infectious c DNA clone; LOM

MeSH Terms

Animals
Antibodies, Viral/blood
Base Sequence
Cell Line
Classical Swine Fever/immunology/*virology
Classical swine fever virus/*genetics/immunology/pathogenicity
Cloning, Molecular
DNA, Complementary/genetics/immunology
Immunization/methods/standards/veterinary
Molecular Sequence Data
Neutralization Tests/veterinary
RNA, Viral/chemistry/genetics
Recombinant Proteins/immunology
Reverse Transcriptase Polymerase Chain Reaction/veterinary
Sequence Analysis, DNA
Specific Pathogen-Free Organisms
Swine
Virulence

Figure

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  Fig. 1 Strategies for construction and in vitro growth characterization of Flc-LOM. (A) Circular map of pFlc-LOM showing the relative positions of the LOM cDNA, ampicillin resistance gene, and P15A origin of replication. Locations of relevant restriction sites and the T7 promoter are indicated. The T7 RNA promoter was located immediately upstream of the 5' end of the genome, and a SrfI site was at the 3' end. The genome sequences are underlined. (B) Using the classical swine fever virus-specific monoclonal antibody LOM01, an indirect immunoperoxidase assay of Flc-LOM was performed in PK-15 cells 3 days after infection with a multiplicity of infection (MOI) of 1 (a and b, × 200). The Flc-LOM biotype was determined using the exaltation of Newcastle disease virus method. Serial dilutions from 101.0 to 106.0 of Flc-LOM were added to primary swine testicular cells. After 4 days, the supernatant was removed and the cells were infected with Newcastle disease virus (104.0 PFU/mL). A cytopathic effect was observed 3 days post-infection and photographed using an inverted microscope (c and d, × 200). (C) Replication kinetics of the Flc-LOM and LOM viruses. PK-15 cells seeded in T25 flasks were infected at a MOI of 1. The data are from three independent experiments. Virus titers are shown as log10 104 median TCID50/mL. ST: swine testicular.

• 

  Fig. 2 Evaluation of clinical responses in pigs infected with Flc-LOM or LOM. CSFV-specific immunity was determined on day 35 p.i. with the virulent CSFV strain SW03. (A) Peripheral white blood cell (WBC) counts in pigs infected with Flc-LOM or LOM. Mean values of the leukocyte counts of the Flc-LOM (n = 5), LOM (n = 5), and control groups (n = 5) are shown. Standard deviations are expressed as error bars (B). Body temperatures were recorded for the Flc-LOM, LOM, and non-vaccinated control groups until 2 weeks after challenge.

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