Fig 1.
Gene map of a full-length antigenomic cDNA (A) of genotype VII NDV strain Banjarmasin/010 (Banj) with fusion protein cleavage site (FPCS) modifications (B). The blue letters indicate amino acid sequences at the FPCS. The arrow indicates the amino acid location where cleavage occurs. The red letters indicate coding nucleotide sequences corresponding to amino acids. The green codon sequences are the original naturally-occurring sequences and the underlined bases indicate the modifications that are introduced in the FPCS to prevent a single nucleotide change generating a basic amino acid residue in the FPCS.
Table 1.
Pathogenicity of rBanj FPCS mutant viruses in 1-day-old SPF chicks.
Fig 2.
Cytopathogenicity of rBanj FPCS mutants in DF-1 cells.
The cells were infected at an MOI of 1 with each of the recombinant viruses. After 3 days, the cytopathic effects (CPE) of each virus infected monolayer was examined under microscope (10X). Ten percent fresh chicken embryo allantoic fluid was used as the source of exogenous protease.
Fig 3.
Plaque morphology of rBanj FPCS mutants in DF-1 cells.
Confluent monolayer of DF-1 cells were infected with each of the recombinant viruses. The infected cells were overlaid with 0.8% methyl cellulose in DMEM, 2% FBS, with or without 10% fresh chicken embryo allantoic fluid as a source of exogenous protease. The viral plaques were obtained after staining with 1% crystal violet.
Fig 4.
F protein cleavage of rBanj FPCS mutants and rLaSota in DF-1 cells.
The cells were infected with respective viruses at an MOI of 1. The cell lysates were collected at 24 h post infection. Western blot was performed using a NDV F cytoplasmic tail anti-peptide rabbit serum. Ten percent fresh chicken embryo allantoic fluid was used as the source of exogenous protease.
Fig 5.
Growth kinetics of rLaSota and rBanj FPCS mutant viruses in DF-1 cells.
Cells were infected at an MOI of 0.001 of each virus and the cell culture supernatant was collected at 8 h intervals for 64 h. All virus titers are expressed as mean log10 TCID50/mL ± SEM (standard error of the mean).
Fig 6.
Growth kinetics of rLaSota and rBanj FPCS mutant viruses in 9-day-old embryonated SPF chicken eggs.
Two hundred μL of 100 TCID50 of each virus in PBS was injected into the allantoic cavity of ten 9-day-old embryonated SPF chicken eggs. Three days after incubation at 37°C, the allantoic fluids was harvested and titrated by hemagglutination (HA) assay. The bars are the means of HA titers. Error bars indicate standard error of the mean.
Fig 7.
Virus titers and tissue tropism of rBanj FPCS mutant viruses in 1-day-old chicks following oculo-nasal inoculation.
Tissue samples from brain, lungs, trachea, and spleen of 3 chickens (n = 3) from each indicated virus group were collected on day 3 post infection, and virus titers were determined by TCID50 assay. The mean virus titers for each tissue sample from 3 chickens are shown. Error bars indicate standard error of the mean.
Fig 8.
Induction of NDV-specific serum antibodies in chickens in response to vaccination with rBanj FPCS mutant viruses, determined by HI (A and B) and serum neutralization assays (C and D). (A and B) Twenty 1-day-old chicks per group were inoculated with 100 μL of (1x105 PFU) virus via oculo-nasal route. Serum samples were collected at 1, 2, and 3 weeks post inoculation. NDV-specific antibodies were determined by hemagglutination inhibition (HI) assay sing 4 HA units of rBanj-LaSota (A) and rLaSota (B). (C and D) The serum neutralizing titers of the vaccinated birds against rBanj-LaSota-eGFP (C) and rLaSota-eGFP (D) viruses are shown. Two-fold serial dilutions of complement-inactivated serum samples were made in a 96-well plate and incubated with 1x103 TCID50 of the recombinant virus at 37°C for 1 h. After incubation at 37°C for 48 h, the cells were washed with PBS and fixed using 4% paraformaldehyde. The fluorescence intensity was measured using a micro plate reader (Tecan, Infinite® M1000) at 490 nm. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction in mean eGFP fluorescence.
Fig 9.
Replication and shedding of vaccine viruses (A) and challenge viruses (B and C) in birds vaccinated at 1-day-old and challenged at 3-week-old, respectively. (A) Chicks in groups of 20 were infected with rBanj FPCS mutants via oculo-nasal route. At day 4 post inoculation (PI), oral and cloacal swabs were collected and analyzed for the presence of virus by inoculation into 9-day-old embryonated SPF chicken eggs. 4 dpi, the allantoic fluid was tested by hemagglutination (HA) assay for the presence of the virus. Shedding of virulent Banjarmasin (Banj) (B) and GB Texas (C) challenge viruses from oral and cloacal swabs. Groups of 10 birds per virus vaccinated with rBanj FPCS mutant viruses at 1-day-old and challenged by the oculo-nasal route with 100 CLD50 virus in 200 μL volume. Oral and cloacal swabs were collected on day 4 post challenge and samples were assayed for virus by inoculation into 9-day-old embryonated chicken eggs and subsequently tested for virus presence by hemagglutination (HA) assay.