Analysis of sequence and haemagglutinin activity of the HN glycoprotein of Newcastle disease virus.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate sequence data for recent Taiwanese strains of Newcastle disease virus (NDV) isolated from 1999 to 2003, covering the full length of the haemagglutinin-neuraminidase (HN) gene and protein. Nucleotide sequence analysis of the HN gene of these recent isolates revealed that the whole HN gene carries an open reading frame encoding 571 amino acids and possesses a shorter C-terminal extension. Six amino acid substitutions in epitopes on the HN glycoprotein of the recent Taiwanese NDV isolates were also found. All the recent Taiwanese NDV isolates have the amino acid sequence (112)RRQKRF(117) for the F protein. A phylogenetic tree analysis based on the nucleotide sequences of the F gene revealed that all recent Taiwanese isolates were related to genotype VII viruses. Since the recent Taiwanese NDV isolates exhibited a low level of haemagglutination (HA) activity, we generated two sets of mutants to elucidate whether mutations in the heptad repeat region of the HN protein could affect the HA activity. To demonstrate the presence of the viruses used in the HA test, a real-time RT-PCR was established to determine the copy number of NDV isolates. From sequence analysis, site-directed mutagenesis, and haemadsorption assays, it was found that the HN glycoprotein of recent Taiwanese NDV isolates carrying a substitution at the amino acid residue 81 (I to M) in the heptad repeat region in the stalk domain showed a dramatic decrease in the activity of HA. We infer from these results that a specific amino acid sequence within the heptad repeat region of the stalk is important for the HA activity of the HN glycoprotein.

Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus.

A robust, ultrasensitive, and accurate quantitative assay was developed for avian reovirus (ARV) with the Light Cycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR). The assay exhibited high specificity as all negative controls and other avian pathogens, such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), and mycoplasma synovia (MS), failed to show any positive detection. A minimum of 39 copies/microl of ARV genomic RNA could be detected by the assay. By dilution analysis, the real-time LC RT-PCR developed in this study was 3-log more sensitive than the conventional RT-PCR for the detection of ARV. The vaccine and field isolates of ARV were detected by the real-time LC RT-PCR. As a result of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of ARV infection.

Genetic and antigenic analysis of Newcastle disease viruses from recent outbreaks in Taiwan.

Portions of the haemagglutinin-neuraminidase (HN) and fusion protein (F) genes of Newcastle disease virus (NDV) isolated from recent outbreaks in Taiwan were amplified and sequenced. These isolates were velogenic, based on the amino acid sequences of the F protein cleavage site and the mean death time in chicken embryos. All the recent viruses contained the amino acid sequences 112RRQKR116 for the C-terminus of the F2 protein. The serological relatedness of recent isolates was determined using a serum neutralization (SN) test. Relatedness values, determined by a cross-SN test, revealed that all belonged to a single serotype but could be classified into distinct subtypes, suggesting that antigenic variations occurred in these isolates. Phylogenetic trees based on the nucleotide sequences of the HN and F genes revealed that recent Taiwanese isolates had evolved into two groups. Antigenic analysis also suggested that there are at least two groups of NDVs involved in recent outbreaks and that these outbreaks in Taiwan might have been caused by co-circulation of multiple velogenic NDV strains.