All experiments involving virulent NDV strains were performed in animal biosafety level-3 facilities.
Cells, viruses and plasmids
Chicken embryo fibroblasts (CEFs) were grown in M199 medium containing 4% fetal calf serum (FCS) and penicillin, streptomycin, and amphotericin. DF1 and BHK-21 cells (clone BSR-T7/5) that express T7 RNA polymerase were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FCS, and BHK-21 cells were used for virus rescue. A reverse genetics-derived genotype VII NDV strain JS5/05  was used as the parental virus. The virus was propagated in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs). The full-length (FL) cDNA clone of JS5/05 and three helper plasmids encoding the NP, P, and L proteins were constructed previously . The plasmid pCMV-C-mOrange2 (Beyotime, Nantong, China) was used as the template to amplify the OFP gene.
Construction of the recombinant NDV cDNA clone containing the OFP gene
The OFP gene was inserted into the genome of JS5/05 using a combined cloning strategy of in-fusion and ligation. Firstly, the pCMV-C-mOrange2 plasmid was used as a template to amplify the OFP gene using the primers (OFP-F and OFP-R). The NDV transcriptional signals, including the gene end, intergenic, and gene start sequences, were added to the upstream of the OFP gene using the forward primer OFP-F. As illustrated in Fig. 1, two gene segments of JS5/05, designated as JS5/05-P and JS5/05-MF, were PCR-amplified using the primer sets (JS5/05-P-F/JS5/05-P-R and JS5/05-MF-F/JS5/05-MF-R), respectively. Then, these fragments were cloned into the linearized pCR2.1 TOPO vector using a Ready-to-Use Seamless Cloning Kit (Sangon, Shanghai, China). After identification by restriction endonuclease digestion and sequencing, the combined gene fragment was transferred to the FL plasmid pNDV/JS5/05 using AgeΙ and BstZ17Ι. The resultant recombinant FL clone was designated as pNDV/JS5/05-OFP. The sequences of primers used for plasmid construction were listed in Table 1 and the detailed information of the primers was also presented.
Rescue of the recombinant NDV
The recombinant NDV was rescued using the reverse genetics as described previously . Briefly, BSR-T7/5 cells were seeded into 35-mm dishes and cultured overnight, and were used for transfection when the confluency reached 70–80%. The cells were washed with PBS for three times, and modified vaccinia virus was added to the cells for adsorption for l h at 37 ℃. After washing with PBS, the full-length cDNA plasmid pNDV/JS5/05-OFP and three helper plasmids were co-transfected into the cells at a ratio of 2: 2: 1: 1 using the X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland). Three days post-transfection, the supernatant was harvested, and 0.2 mL was inoculated into the allantoic cavities of 9-day-old ECEs. Four days post inoculation (pi), the allantoic fluids were harvested, and the presence of virus was identified by hemagglutination (HA) test. The HA-positive allantoic fluids were propagated in 9-day-old ECEs. The recovered virus was designated as rJS-OFP.
Characterization of the recombinant NDV
To determine the genetic stability of the recombinant NDV, the virus was propagated in ECEs for five passages, and the presence of the OFP gene in NDV genome was detected using RT-PCR. Virus yield was analyzed by measuring HA activity, 50% tissue culture infectious dose (TCID50) in CEF cells, and 50% embryo infectious dose (EID50). In addition, to assess the impact of OFP gene insertion on NDV replication, the growth kinetics of the recombinant virus and parental virus was determined in CEF cells. In brief, the cells were seeded in six-well plates and cultured in the complete medium (M199 medium containing 4% FCS) overnight. The cells were then inoculated with the viruses at a multiplicity of infection (MOI) of 0.01. After virus absorption for 1 h, the inoculum was removed, and the maintaining medium (M199 medium supplemented with 1% FCS) was added. The culture supernatants were harvested at 24, 36, 48, 60, and 72 h pi, and the virus content in the supernatants was determined. The collected supernatants were tenfold serially diluted in the maintaining medium and inoculated into CEFs. At 72 h pi, the presence of the virus in the culture supernatants was detected using HA assay and TCID50 titers were calculated. Additionally, the virulence of the recombinant virus was evaluated by standard MDT test . Briefly, the recombinant and parental virus were serially diluted and 0.1 mL of the 10–6 to 10–10 dilutions was inoculated into 9-day-old ECEs, five eggs per dilution. The eggs were monitored three times a day for 5 days. The death time of each chicken embryo was recorded and the MDT was calculated.
Expression of the OFP by the recombinant NDV
To detect the expression of the OFP protein, 1 × 105 of DF1 cells in a 12-well plate were inoculated with the recombinant virus and parental virus at a MOI of 1. At 36 h pi, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then incubated with a monoclonal antibody against the NDV HN protein as the primary antibody at 37 ℃ for 1 h. The cells were then incubated with FITC-conjugated goat anti-mouse antibody (TransGen Biotech, Beijing, China) as the secondary antibody at 37 ℃ for 1 h. Cell nuclei were stained with DAPI (Beyotime). The cells were examined under a fluorescence microscope to monitor the expression of the OFP and NDV HN protein.
Pathogenicity of the recombinant NDV in chickens
To assess the pathogenicity of the recombinant virus in vivo, twenty-seven 5-week-old SPF white leghorn chickens were randomly divided into three groups of nine chickens per group. Chickens were inoculated with 105 EID50 of the recombinant virus and parental virus by the intranasal and intraocular routes. The animals were monitored daily and clinical signs were scored as following: 0, normal; 1, mild symptoms (mild depression); 2, moderate symptoms (diarrhea, respiratory signs); 3, severe symptoms (marked depression, open-mouth breathing, paralysis and obvious neurological signs); 4, dead. At day 1 and 3 pi, three chickens per group were euthanatized for observation of gross pathology. In addition, the spleen, lung, and intestinal tract were collected for virus titration. The remaining three chickens of each group were observed daily for 12 days for clinical signs and mortality.