Fig 1.
Baculovirus construct for production of chimeric virus-like particles.
(A) construction of a F/HA fusion gene containing ectodomain of the NDV KR-005/00 strain F gene and TM/CT domain of the H5N1 avian influenza virus ES03 strain HA gene. The schematic diagram shows the location of the ectodomain, transmembrane (TM) domain, and cytoplasmic tail (CT) domain of the F and HA genes and their junction. The TM/CT domain of the HA gene was fused to the carboxyl terminal of the F gene to form the full-length F/HA fusion gene; (B) HA, M1, and chimeric F/HA genes with its own polyhedrin promoter (Pph) and transcription termination sequences are indicated.
Table 1.
Primer sets used for RT-PCR amplification of genes of HPAIV ES03 and NDV KR-005/00.
Fig 2.
Analysis of concentrated chimeric VLP: (A) Western blot identification of the HA protein using anti-H5 monoclonal antibody. M, standard molecular size marker (EBM-1032, Elpisbio), HA, chimeric VLPs; (B) Western blot identification of the F/HA chimeric protein using anti-NDV F monoclonal antibody; (C) Western blot identification of the M1 protein using anti-H5N1 anti-serum; (D) TEM identification of chimeric VLP with scale bar; (E) immunogold-labelled chimeric VLP: VLP were probed with anti-H5 monoclonal antibody, counterstained with 10nm gold spheres coupled to anti-mouse IgG and anti-NDV anti-serum, counterstained with 25nm gold spheres coupled to anti-chicken IgG.
Fig 3.
HPAIV Experiments in SPF chickens.
(A) HI titers after three weeks post vaccination and two weeks after HPAIV challenge: serum samples were analyzed with the standard HI test for the presence of HI antibodies against ES03 H5 AIV. Error bars represent standard deviations. ***, P < 0.001, **, P < 0.01, (B) Survival curve after lethal HPAIV challenge. Three weeks after immunization, SPF chickens were intranasally infected with a lethal dose of ES03 HPAI virus. The chickens were observed daily for a period of 14 days for survival. (C) Differentiation of vaccinated chickens from vaccinated and infected chickens. Serum samples were taken before challenge (3 weeks post-vaccination) and 14 days post challenge. NP antibody levels were tested with a commercially available NP-cELISA kit. Each dot represents the NP-specific antibody value of each chicken.
Fig 4.
NDV Experiments in SPF chickens.
(A) Mean levels of antibody against NDV in serum. Levels of antibody against NDV in serum were determined using a commercially available ELISA kit after three weeks post vaccination. The cut-off value was presented as dot line. Statistical significance was determined by ANOVA with the Tukey-Kramer post hoc test. ***, P < 0.001; **, P < 0.01 (B) Survival curve after lethal NDV challenge. Three weeks after immunization, SPF chickens were intramuscularly infected with a lethal dose of KR-005/00 strain NDV. The chickens were observed daily for a period of 14 days for survival. (C) HI titers after two weeks post challenge for DIVA: Serum samples were analyzed with the standard HI test for the presence of HI antibodies against the La Sota NDV strain. Error bars represent standard deviations. ***, P < 0.001,
Table 2.
Challenge HPAI virus shedding from oropharyngeal swab samples.
Table 3.
Challenge HPAI virus shedding from cloacal swab samples.