Generating Chimeric NDV Virus Clones

The full-length infectious cDNA clones (ICs) of the NDV heat-resistant strain HR09 and thermolabile strain La Sota, named cHR and cLa, respectively, were generated in plasmid TVT-7R (0.0) (Johnson et al., 2000 (link)) from six fragments that were amplified by PCR using high-fidelity DNA polymerase (New England BioLabs, United States) as described in our previous research (Ruan et al., 2020 (link)).
Three chimeric ICs, cHR-La-F, cHR-La-HN, and cHR-La-F/HN, were constructed by replacing the open reading frames (ORFs) of the F and/or HN genes in the cHR strain with the counterpart fragments from the La Sota strain (Figure 1). Briefly, the ORFs of the F and/or HN genes were amplified by PCR and then inserted into the corresponding sites in full-length ICs of La Sota according to the HiFi DNA assembly cloning method (New England BioLabs, United States). Similarly, ICs of the chimeric viruses cLa-HR-F, cLa-HR-HN and cLa-HR-F/HN were generated by replacing the ORFs of the F and/or HN genes in cLa with the corresponding fragments from cHR (Figure 1).