Figure 1. Schematic strategy for the construction of two chimeric NDVs. The F gene of genotype VIII thermostable HR09 strain was replaced with the mutated F gene of genotype VII DT-2014 strain.

Figure 1. Schematic strategy for the construction of two chimeric NDVs. The F gene of genotype VIII thermostable HR09 strain was replaced with the mutated F gene of genotype VII DT-2014 strain.

Figure 2. Construction and validation of the chimeric NDV rcHR09-CI and rcHR09-CII. The total RNA of rescued viruses was extracted and reverse-transcribed into cDNA, the F gene of rcHR09-CI (A) and rcHR09-CII (B) was amplified and set to sequencing. The full length of the chimeric NDV rcHR09-CI (C) and rcHR09-CII (D) was amplified using 10 pairs of primers, and S1–S10 are the representation of them.

Figure 2. Construction and validation of the chimeric NDV rcHR09-CI and rcHR09-CII. The total RNA of rescued viruses was extracted and reverse-transcribed into cDNA, the F gene of rcHR09-CI (A) and rcHR09-CII (B) was amplified and set to sequencing. The full length of the chimeric NDV rcHR09-CI (C) and rcHR09-CII (D) was amplified using 10 pairs of primers, and S1–S10 are the representation of them.

Figure 3. The biological characteristics of the chimeric NDV rcHR09-CI and rcHR09-CII. (A) The two chimeric NDV rcHR09-CI and rcHR09-CII have similar growth kinetic with their maternal strain HR09. The growth curve of the two chimeric viruses and their maternal strain HR09 were measured with EID50 method in 10-day-old SPF embryonated chicken eggs. (B) The two chimeric NDV rcHR09-CI and rcHR09-CII share the thermostability with HR09. Total 108 EID50 of the NDV rcHR09-CI, rcHR09-CII, HR09, and La Sota strains were incubated in 56 °C for 10-, 20-, 30-, 40-, 50-, and 60-min, respectively. Then, the titers of the four NDVs were measured using EID50 method.

Figure 3. The biological characteristics of the chimeric NDV rcHR09-CI and rcHR09-CII. (A) The two chimeric NDV rcHR09-CI and rcHR09-CII have similar growth kinetic with their maternal strain HR09. The growth curve of the two chimeric viruses and their maternal strain HR09 were measured with EID50 method in 10-day-old SPF embryonated chicken eggs. (B) The two chimeric NDV rcHR09-CI and rcHR09-CII share the thermostability with HR09. Total 108 EID50 of the NDV rcHR09-CI, rcHR09-CII, HR09, and La Sota strains were incubated in 56 °C for 10-, 20-, 30-, 40-, 50-, and 60-min, respectively. Then, the titers of the four NDVs were measured using EID50 method.

Figure 4. The chimeric viruses induced a high level of antibody response. The SPF chickens were immunized with 106 EID50 rcHR09-CI, rcHR09-CII and the commercial vaccine La Sota strain through oculonasal route, respectively. The serum was collected at 1-, 2-, and 3-weeks post immunization, the antibody titer was measured using HI method against themselves (A) and the cross HI antibody titer against ZJ1 (B). (ns: no significant difference; * p < 0.05; ** p < 0.01).

Figure 4. The chimeric viruses induced a high level of antibody response. The SPF chickens were immunized with 106 EID50 rcHR09-CI, rcHR09-CII and the commercial vaccine La Sota strain through oculonasal route, respectively. The serum was collected at 1-, 2-, and 3-weeks post immunization, the antibody titer was measured using HI method against themselves (A) and the cross HI antibody titer against ZJ1 (B). (ns: no significant difference; * p < 0.05; ** p < 0.01).

Figure 5. QRT-PCR quantitation of NDV ZJ1 shedding in challenged chickens. The chickens were challenged with 106 EID50 NDV genotype VII ZJ1 strain at 21 days post immunization. The cloacal swab samples (A,C) and oropharyngeal swab samples (B,D) were collected at the 3- and 5-days post challenge, and the levels of NDV ZJ viral shedding was quantitated via QRT-PCR. Values were shown as mean values ± standard deviation (mean ± SD) in each group. (ns: no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5. QRT-PCR quantitation of NDV ZJ1 shedding in challenged chickens. The chickens were challenged with 106 EID50 NDV genotype VII ZJ1 strain at 21 days post immunization. The cloacal swab samples (A,C) and oropharyngeal swab samples (B,D) were collected at the 3- and 5-days post challenge, and the levels of NDV ZJ viral shedding was quantitated via QRT-PCR. Values were shown as mean values ± standard deviation (mean ± SD) in each group. (ns: no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6. Chimeric NDV rcHR09-CI and rcHR09-CII provide full protection against the challenge of ZJ1. At 3 weeks post immunization, the chickens were challenged with 106 EID50 ZJ1 strain. The survival status was monitored for a total of 14 days.

Figure 6. Chimeric NDV rcHR09-CI and rcHR09-CII provide full protection against the challenge of ZJ1. At 3 weeks post immunization, the chickens were challenged with 106 EID50 ZJ1 strain. The survival status was monitored for a total of 14 days.

Table 1. The pathogenicity and stability evaluation of the two chimeric NDVs (total 20 passages).

Table 1. The pathogenicity and stability evaluation of the two chimeric NDVs (total 20 passages).