The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.
The nucleocapsid (NP) and phospho-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilised on a solid phase and then interacted with radio-labelled [(35)S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using this approach, a highly interactive region was identified to be resided at the first 25 amino acids of NP N-terminus. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end of NP. However, it is possible that the 49 amino acids at the C-terminal end might have another contact region for P protein, which is not as critical as the N-terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P.
A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.
The objective of this work was to study the effect of storage temperatures and duration on the stability of fermented red dragon fruit drink (FRDFD) on its betacyanins content, physicochemical and microbiological qualities (BPM) and determining sensory acceptability. Results showed that both storage temperatures and duration have a significant effect on betacyanins content and physicochemical properties of FRDFD. Aerobic mesophilic and yeast and mold counts were lower than 1 × 103 CFU/mL for FRDFD stored at both temperatures. The loss of betanin (16.53-13.93 g/L) at 4 °C was 15.73% with no significant changes in physicochemical properties from week two onwards compared to 56.32% (16.53-7.22 g/L) of betanin loss at 25 °C. At week eight, FRDFD stored at 4 °C still contained 13.93 g/L betanin with a pH value of 3.46, suggested its potential as a functional drink which is sensory acceptable (mean score > 80% using hedonic test) among consumers.