PCR-based reverse genetics strategy for bluetongue virus recovery

Area of Science:

• Virology
• Molecular Biology
• Animal Health

Background:

• Bluetongue virus (BTV) is an emerging, insect-borne pathogen impacting livestock and wild ruminants, causing significant agricultural economic losses.
• BTV possesses a 10-segment double-stranded RNA genome, and its rescue via traditional reverse genetics (RG) involves laborious in vitro RNA synthesis from cDNA templates.
• Current BTV RG methods are complicated by expensive reagents, meticulous procedures, and the need for specific cloning vectors.

Purpose of the Study:

• To develop a simplified and efficient PCR-based reverse genetics (RG) system for Bluetongue virus (BTV) rescue.
• To establish a convenient platform for BTV research as an alternative to existing methods.
• To explore the applicability of this RG strategy for other Orbiviruses.

Main Methods:

• Development of a PCR-based reverse genetics (RG) system for BTV.
• Utilized a two-step transfection process involving helper plasmids and PCR products of viral genome segments.
• Characterization of rescued viruses using indirect immunofluorescence assays (IFA) and gene sequencing.

Main Results:

• Successfully rescued viable BTV using the novel PCR-based RG system after two transfection steps.
• Characterization confirmed the identity and integrity of the rescued viruses.
• Comparative analysis revealed that reassortant BTV containing specific genome segment 2 variants exhibited slightly slower proliferation compared to wild-type BTV.

Conclusions:

• A convenient and efficient PCR-based RG platform for BTV has been established.
• This new strategy serves as a viable alternative to traditional BTV rescue methods.
• The developed RG strategy shows potential applicability for rescuing other viruses within the Orbivirus genus.