The Korean viscerotropic velogenic (vv) NDV Kr-005/00 strain and the lentogenic La Sota strain of NDV were grown in 10-day-old specific pathogen free (SPF) embryonated chicken eggs. The Autographa californica nucleopolyhedrovirus (AcNPV) and recombinant baculoviruses were grown and assayed in either Spodoptera frugiperda 21 (Sf21) cells or Trichoplusia ni High Five (Hi5) cells in Grace's medium (GibcoBRL, USA) supplemented with 10% fetal bovine serum. The virus re-isolation of the challenge virus was performed in primary chicken embryo fibroblast (CEF) cell cultures.

Viral RNA was extracted directly from the infective allantoic fluid using an RNeasy kit (Qiagen, USA) according to the manufacturer's recommendations. Four paired primers were used to amplify the complete coding sequence of the fusion (F) and Hemagglutination-Neuraminidase (HN) genes of the La Sota and Kr-005/00 strains (primer sequences, Table 1).

The purified reverse transcription-polymerase chain reaction products of each gene were cloned into a cloning vector, pCR 2.1 (Invitrogen, USA). To construct the transfer vectors pBacF(V) and pBacHN(V), the F and HN genes of the Kr-005/00 strain were excised by digestion with EcoR I and inserted into the EcoR I site of pBacPAK9 (Clontech, USA). The F gene of the La Sota strain was excised by digestion with Xho I and BamH I, and the HN gene of the La Sota strain was excised by digestion with Not I and BamH I. The excised fragments were repaired with T4 polymerase and inserted into the Sma I site of pBacPAK9. The resulting transfer vectors were pBacF(L) and pBacHN(L). All transfer vectors contained the upstream polyhedron promoter of the inserted gene and the downstream polyadenylation sequences. The orientation of the inserted gene was examined by a restriction enzyme treatment and by sequencing the 5' junction region.

Sf21 cells were transfected with 0.5 µg of the transfer vector plasmid DNA (four each) and BacPAK6 viral DNA (Bsu36 I digest) using the BacPAK baculovirus expression system (Clontech, USA) according to the manufacturer's instructions. After 72 h incubation at 27℃, the culture supernatants were subjected to a plaque assay to isolate the recombinant viruses. The selected plaque was examined to confirm that the target gene had been inserted into the viral genome using polymerase chain reaction with the Bac1 and Bac2 primers (Clontech, USA). Four recombinant viruses, rND F induced from vvNDV rNDF(V), rND F from La Sota strain rNDF(L), rND HN from vvNDV rNDHN(V), and rND HN from La Sota rNDHN(L), were prepared using pBacF(V), pBacF(L), pBacHN(V), and pBacHN(L), respectively. Recombinant viruses were stored at 4℃ until needed.

An immunofluorescence assay was performed without prior fixation, as described previously [22]. Briefly, the Sf21 cells were infected with the recombinant at an MOI of 5 and incubated at 27℃ for 48 h. The unfixed infected cells were washed with phosphate-buffered saline (PBS) and incubated with polyclonal chicken anti-NDV sera or monoclonal antibodies specific to the NDV HN glycoprotein for 1 h at room temperature. After washing with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated anti-chicken or anti-mouse immuno-globulin (Cappel, USA) for 1 h at room temperature. The cells were washed again with PBS and examined for fluorescence.

All chickens were derived from SPF eggs of White Leghorn parents (Lohmann, Germany). The animals were housed and reared in positive-pressure isolators until they were immunized.

Sf21 cells infected with recombinant viruses were sonicated with Soniprep 150 (Sanyo, UK) at 18 Khz for 5 min, clarified by centrifugation, mixed thoroughly using an Omnimixer with Montanide Incomplete Seppic Adjuvant (ISA-70; Seppic, France) at a ratio of 3 : 7, and used as antigens for immunization. For vaccination, chickens were inoculated intramuscularly with the Seppic-adjuvanted antigen (detailed doses are described in each Table). Each group of vaccinated and unvaccinated chickens was challenged with the vvNDV Kr-005/00 strain through an intraocular inoculation with an 10^5.5 EID₅₀ per bird. vvNDV Kr-005/00 was the representative strain of genotype VII, which is the dominant epizootic genotype in Korea [11]. The chickens were kept under observation for 14 days after infection.

To examine the protective effect of each recombinant glycoprotein, the SPF chickens were immunized with infected cells containing rNDF(V), rNDF(L), rNDHN(V), or rNDHN(L) and the Seppic-adjuvant. In Experiment 1, fifty-five 5-week-old chickens were divided into 11 groups, in which rNDF(V) and rNDHN(V) were subdivided into 4 groups based on the inoculum dose. Five chickens from each group were housed in separate isolators. All chickens were immunized twice at 3-week intervals and challenged 3 weeks after the second immunization.

In Experiment 2, 120 3-week-old chickens were divided into 8 groups. Four groups were immunized with 1 × 10⁶ cells of the infected lysate with an adjuvant of rNDF(V), rNDF(L), rNDHN(V), or rNDHN(L). Two groups were immunized with a half dose (0.5 × 10⁶ cells) of rNDF(V) and rNDHN(V), and the last group was immunized with the combined rNDF(V) and rNDHN(V)-infected cells. The remaining group was used as the non-immunized control. Fifteen chickens in each group were housed in separated isolators. Three weeks after the first immunization, seven chickens from each group were challenged to evaluate the level of immunity, and the remaining eight chickens were given a booster immunization with the same antigen. Three weeks after the second immunization, the chickens were challenged. The protective effect was evaluated by observing the chickens for any clinical signs and mortality. The re-isolation of the challenge virus was carried out in CEF cells with the oropharyngeal and cloacal swab samples 5 day after the challenge infection. All chickens were sampled at 5 d post infection (dpi) including three dead chickens at 4 dpi, one chicken from the rND F(V) group and two chickens from the control group, after the 1st immunization in Experiment 2 (Table 3). For virus isolation, the swab samples were suspended in 1 ml of cell culture medium with antibiotics. And then the clarified supernatants were titrated for virus infectivity in CEF cells.

The Hemagglutination inhibition (HI) test was performed as described in the OIE manual of standard diagnostic tests [23]. The level of agglutination was assessed by titling the plates. Only those wells in which the RBCs streamed at the same rate as the control wells were considered to exhibit hemagglutination inhibition. ELISA was performed using the Newcastle disease antibody detection kit (IDEXX Laboratories, USA) according to manufacturer's instructions.

For statistical analyses, the two-tailed Fisher's exact test and the Student's t-test were used for the protective immunity and antibody response, respectively. A p-value <0.05 was considered significant.