Figure 1.
Phylogenetic analysis based on the nucleotide (nt) sequences of complete F open reading frame of Indonesian strains in all genotypes of NDV.
The tree was constructed by bootstrap analysis (1000 replications) using the neighbor-joining of the Kimura-2-parameter method for nt differences in the MEGA 4.0 phylogenetic analysis program. Scale bar shows number of base substitutions per site. Bootstrap values are shown at the nodes.
Table 1.
Database of NDV Indonesia isolates in this study.
Table 2.
Identities of amino acid sequences of F protein.
Table 3.
Identities of amino acid sequences of HN protein.
Figure 2.
Construction of a full-length antigenomic cDNA of NDV Indonesia strain Ban/010 and modification of its F protein cleavage site.
(A) Construction of the antigenomic cDNA. Eight cDNA fragments were generated from viral RNA by RT-PCR using primers that inserted seven unique restriction sites (sequence positions are indicated). Using these sites, the cDNA fragments were inserted sequentially into the pBR322/dr plasmid vector, flanked on the upstream side by a T7 RNA polymerase promoter sequence and on the downstream side by the hepatitis delta virus ribozyme sequence (Rbz) followed by a T7 terminator sequence (Tm). (B) Modification of the F protein cleavage site. The sequence of the cleavage site of the wt Ban/010 virus is shown, as well as the altered cleavage site in mutant Ban/AF. The mutated residues and nucleotides are shown in bold. The arrow indicates where cleavage occurs.
Table 4.
Pathogenicity and stability of the Ban/AF virus.
Figure 3.
Syncytia and plaque formation by NDV strains in DF1 cells.
(A) DF1 monolayers were infected with NDV strains B1, LaSota, Ban/010 and Ban/AF at an MOI of 0.01. The infected cells were cultured in the presence or absence of 10% normal allantoic fluids as exogenous protease. The cells were photographed at 36 hpi. (B) The cells were infected with the indicated viruses at an MOI of 0.01. The infected cells were overlaid by 0.8% methylcellulose DMEM and cultured in the presence or absence of 10% normal allantoic fluid for 7 days. The cells was fixed and stained with 1% crystal violet.
Figure 4.
Effect of exogenous protease on the growth and F protein cleavage of the Ban/AF virus in DF1 cells.
(A) Cells were infected with the mutant Ban/AF at an MOI of 1 and 0.01, and cultured in the presence or absence of 10% normal allantoic fluid. 100-ul aliquots of culture supernatants were collected daily for titration of viruses by the TCID50 assay. (B) Cells were infected with a 10-fold dilution series of allantoic fluid from Ban/010-infected eggs, and cultured in the presence or absence of 10% normal allantoic fluids. The cells were fixed and immunostained using anti-NDV N protein antiserum (20), followed by incubation with secondary antibody and detection by substrate AEC plus chromogen (Dako, USA). (C) Cleavage of the Ban/AF and B1 F proteins in infected DF1 cells. The cells were infected with viruses at an MOI of 1. The cell lysates were collected at 6, 12 and 24 h post infection. Western-blot was performed with an anti-NDV F cytoplasmic tail peptide rabbit serum.
Figure 5.
Replication and shedding of vaccine viruses Ban/AF, B1, and LaSota in 1-day old chicks.
Chicks in groups of 3 per virus were infected with virus by a single nasal drop each, as described in Materials and Methods. At 4, 6, and 8 dpi, three birds per virus per day were sacrificed, and tissue from the indicated organs, as well as oral and cloaca swabs, were collected. These were assayed for the presence of virus by inoculation into 9-day embryonated chicken eggs, as summarized in Table 2. Samples that were positive in Table 5 were analyzed to determine virus titers by TCID50 assay. Samples: lung (A), trachea (B), spleen (C), pancreas (D), oral swabs (E) and cloaca swabs (F).
Table 5.
vaccine virus replication and shedding in 1-day old chicks.
Figure 6.
Replication and shedding of vaccine viruses Ban/AF, B1 and LaSota in 2-week-old chickens.
Chickens in groups of 3 were infected with virus by a single nasal drop each, as described in Materials and Methods. At 4, 6, and 8 dpi, three birds per virus per day were sacrificed, and tissue from the indicated organs, as well as oral and cloaca swabs, were collected. These were assayed for the presence of virus by inoculation into 9-day embryonated chicken eggs, as summarized in Table 6. Samples that were positive in Table 3 were analyzed to determine virus titers by TCID50 assay. Samples: lung, trachea, spleen, and pancreas (A), oral swabs (B), cloaca swabs (C).
Table 6.
Vaccine virus replication and shedding in 2-week-old chickens.
Figure 7.
Protective efficacy of vaccine viruses Ban/AF and B1 in 1-day-old chicks against challenge with virulent strains
Ban/010 and GB Texas. Chicks were inoculated with vaccine viruses Ban/AF or B1 by a single nasal drop each, or were mock-infected, as described in Materials and Methods. At 21 dpi, birds from each group were challenged with 100 LD50 of the Ban/010 or GB Texas viruses by the oculonasal route. The birds were observed daily for survival. Each challenge group had 10 birds except for the 1-day-old chicks immunized with Ban/AF, which had 12 birds in the Ban/010 challenge group and 11 birds in the GB Texas challenge group.
Figure 8.
Shedding of Ban/010 or GB Texas challenge virus in 1-day-old chicks previously immunized with Ban/AF or B1.
From the experiment shown in Fig. 7, oral and cloaca swabs were collected at 4, 6 and 8 days post-challenge, and samples were assayed for virus detection by inoculation into 9-day embryonated chicken egg. Samples that were positive in Table 7 were titrated by TCID50 assay. Note that virus was detected only in B1-vacciniated chicks.
Table 7.
Viral shedding of vaccinated 1-day old birds after challenge.