Rescuing Of Porcine Circovirus 3 And 4 Via Reverse Genetics And Quantum Dot Labeling

Porcine circovirus(PCV)is a non-enveloped DNA virus with a diameter of approximately 20 nanometers.It belongs to the Circovirus genus of the Circoviridae family.PCV comprises four subtypes,PCV1 to PCV4,among which PCV2 is the most significant pathogen causing porcine circovirus disease(PCVD/PCVAD)and has caused serious economic impacts on the global swine industry.Although the pathogenicity of PCV3 and PCV4 remains controversial,their infection rates in pig populations have gradually increased and they have become PCV subtypes of concern.Traditional virus labeling methods require the use of fluorescent dyes or antibodies,but these methods have many drawbacks such as low fluorescence intensity,poor stability,and low photothermal conversion efficiency.In recent years,quantum dots have become a new type of virus label due to their unique physical properties and high fluorescence quantum yield.The fluorescence wavelength of quantum dots is tunable,and they can be excited to emit high fluorescence intensity signals,thus achieving high sensitivity and specificity detection of viruses.Therefore,the use of quantum dots as virus labels has become one of the hot topics in current virology research.Based on the aforementioned situation,this article focuses on reverse genetics technology and carries out the following series of work.1.Rescue of PCV4 and studies on its infection in piglets’ bodies.To rescue PCV4,the full-length sequence MT311854.1 from the NCBI database was used as a template to construct the recombinant plasmid p SK-PCV4.Transfection of p SK-PCV4 into PK-15 cells resulted in the expression of PCV4,as confirmed by immunoblot analysis.The rescued PCV4 virus maintained stable copy numbers during blind passage up to the fourth generation,indicating its ability for infection and propagation.Western blot and immunofluorescence assays confirmed successful expression of the PCV4 capsid protein(Cap)in multiple passages.Transmission electron microscopy revealed the presence of mature PCV4 viral particles,approximately 20 nm in diameter,validating the correct assembly process of the rescued virus generated from the p SK-PCV4 infectious clone.2.Rescue of PCV3 and viral labeling.Using the reported full-length sequence MF318451.1 from the NCBI database,recombinant plasmids p SK-PCV3 and p SK-PCV3-Avi were constructed.The Avi tag sequence(GLNDIFEAQKIEWHE)was inserted at the N-terminus of the ORF2 region,which encodes the Cap protein.Models confirmed Avi peptide expression on the surface of PCV3 and PCV3-Avi viral particles.Transfection of infectious clones into PK-15 cells successfully rescued PCV3 and PCV3-Avi,as confirmed by Western blot analysis.Blind passage of the rescued viruses maintained stable copy numbers.Western blot and immunofluorescence assays in the P4 generation confirmed normal Cap protein expression and showed no interference from the Avi tag.Electron microscopy confirmed the presence of mature viral particles.Avi labeling with biotin and streptavidinconjugated quantum dots allowed successful co-localization with PCV3-Avi in cellular assays.Subcutaneous injection of quantum dot-labeled PCV3-Avi in mice demonstrated viral diffusion,enabling the study of PCV3 pathogenic mechanisms.In summary,the study one successfully constructed the infectious clone p SK-PCV4 and rescued the PCV4 virus by transfecting it into PK-15 cells.The virus was found to replicate effectively and exhibit expected biological characteristics,including the ability to infect multiple tissues in piglets and cause certain pathogenicity,as well as to induce significant immune responses in vivo.The study two successfully constructed the infectious clones PCV3 and PCV3-Avi,rescued PCV3 and PCV3-Avi viruses,and performed biological characterization tests,which confirmed that there were no biological differences between the two viruses.The study also found that quantum dots could effectively label PCV3-Avi for virus tracking analysis.