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<p>Classical Swine Fever (CSF) is one of the most important viral infections affecting swine. In the present study, the complete genome of the cell culture-adapted lapinized Indian Classical Swine Fever virus (CSFV) was cloned into the pBR322 vector using an improved method. The viral RNA was amplified by RT-LA PCR into two overlapping fragments of ~ 6.5&#xa0;kb and&#xa0;5.8&#xa0;kb sizes, and cloned separately into the pTZ57R/T vector. To generate CSFV full-length cDNA, the segments were fused utilising a restriction endonuclease region located within the overlapping region. The complete genome was then cloned into the pBR322 vector and designated as pBR/T7-CSFV. The recombinant plasmid was confirmed by PCR, restriction enzyme analysis, and nucleotide sequencing. The full-length cDNA backbone of CSFV can serve as a reverse genetics model with the T7 promoter and can be used for creating recombinant viruses and for developing a DIVA strategy for CSFV. This study describes a modified method for full-length amplification of an Indian CSFV isolate and cloning it into the pBR322 vector.</p> Graphical Abstract <p></p>

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Construction of full length cDNA clone of classical swine fever virus using an improved strategy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月21日

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  • Parveen Kumar,
  • Pronab Dhar,
  • Vikramadiya Upmanyu,
  • Rekha Yadav,
  • Amit Kumar,
  • Sanjeevna Kumari Minhas,
  • Ashok Kumar Tiwari

摘要

Classical Swine Fever (CSF) is one of the most important viral infections affecting swine. In the present study, the complete genome of the cell culture-adapted lapinized Indian Classical Swine Fever virus (CSFV) was cloned into the pBR322 vector using an improved method. The viral RNA was amplified by RT-LA PCR into two overlapping fragments of ~ 6.5 kb and 5.8 kb sizes, and cloned separately into the pTZ57R/T vector. To generate CSFV full-length cDNA, the segments were fused utilising a restriction endonuclease region located within the overlapping region. The complete genome was then cloned into the pBR322 vector and designated as pBR/T7-CSFV. The recombinant plasmid was confirmed by PCR, restriction enzyme analysis, and nucleotide sequencing. The full-length cDNA backbone of CSFV can serve as a reverse genetics model with the T7 promoter and can be used for creating recombinant viruses and for developing a DIVA strategy for CSFV. This study describes a modified method for full-length amplification of an Indian CSFV isolate and cloning it into the pBR322 vector.

Graphical Abstract