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Newcastle disease virus in pakistan: genetic characterization and implication in molecular diagnosis.

ABSTRACT: Newcastle disease (ND) is a fatal and contagious disease that poses a constant threat to the poultry industry around the globe. Due to the complex clinico-pathological picture and high genetic variability, the efficient diagnosis of NDV strains is a challenge. In an emerging wave of ND in the north of Pakistan, samples from six outbreaks in commercial poultry and two from healthy backyard poultry flocks were screened for NDV. A real-time PCR based on the fusion and polymerase genes of NDV detected all six isolates whereas a validated real-time PCR based on the matrix gene failed to detect any of these isolates, most likely due to substantial mismatches in the probe-binding site. All isolates have shown ICPI and MDT values similar to the velogenic form of NDV strains. The cleavage site in the F protein was found to be (112)RRQKR?F(117), typical of virulent NDV. Phylogenetic reconstruction, based on fusion and matrix genes, provided enough evidences to consider these isolates as a new subgenotype within genotype VII. This study raised concerns about the genetic variability of NDV circulating in Pakistan, and sensitivity of the assays for the detection of the NDV isolates in clinical samples.

SUBMITTER: Munir M

PROVIDER: S-EPMC3550779 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Newcastle disease virus in pakistan: genetic characterization and implication in molecular diagnosis.

Munir M M Munir M M Zohari S S Berg M M

Indian journal of virology : an official organ of Indian Virological Society 20120704 3

Newcastle disease (ND) is a fatal and contagious disease that poses a constant threat to the poultry industry around the globe. Due to the complex clinico-pathological picture and high genetic variability, the efficient diagnosis of NDV strains is a challenge. In an emerging wave of ND in the north of Pakistan, samples from six outbreaks in commercial poultry and two from healthy backyard poultry flocks were screened for NDV. A real-time PCR based on the fusion and polymerase genes of NDV detect ...[more]

PMID: 24293827

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Project description:Newcastle disease (ND) is a highly fatal, infectious, viral disease, and despite immunization with live and inactivated vaccines, the disease is still endemic, causing heavy morbidity and mortality leading to huge economic losses to the poultry industry in Pakistan. Therefore, the present study was aimed for the first time in the country at using novel virosomal technology to develop the ND vaccine using an indigenous highly virulent strain of the virus. ND virosome was prepared using Triton X-100, and SM2 Bio-Beads were used to remove the detergent and reconstitute the viral membrane into virosome. Confirmation was done by transmission electron microscopy and protein analysis by SDS-PAGE. In vitro cell adhesion property was observed by incorporating green fluorescent protein (GFP), producing plasmid into virosome and in vitro cell culture assay. Sterility, safety, and stability of the vaccine were tested before in vivo evaluation of immunogenicity and challenge protection study in commercial broiler. The virosome vaccine was administered (30 μg/bird) at days 7 and 14 through the intranasal route in comparison with commercially available live and inactivated ND vaccines. Results revealed significantly high (p < 0.05) and clinically protective hemagglutination inhibition (HI) antibody titers at 7, 14, 21, and 28 days postimmunization with the virosome vaccine in comparison to the negative control. The GMTs were comparable to live and inactivated vaccines with nonsignificant (p > 0.05) differences throughout the experiment. Antibody levels increased in all vaccinated groups gradually from the 7th day and were maximum at 28th-day postvaccination. In the virosome-administered group, GMT was 83.18 and 77.62 at 21st and 28th-days postvaccination, respectively. Challenge revealed 100%, 90%, and 80% protection in virosome, live, and inactivated vaccinated groups, respectively. Under given experimental conditions, we can conclude that ND virosome vaccine prepared from the indigenous virus was found to be safe and immunogenic.

Project description:BackgroundNewcastle disease (ND) is a devastating worldwide disease of poultry characterized by increased respiration, circulatory disturbances, hemorrhagic enteritis, and nervous signs. Sequence analysis shows several amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of recent Shaanxi strains. Both Cross protection and cross serum neutralization tests revealed that the traditional vaccine strains were unable to provide full protection for the flocks.MethodsTo better understand the epidemiology of Newcastle disease outbreak, a portion of the F gene and the full-length HN gene were amplified from Shaanxi isolates by reverse transcription-polymerase chain reaction (RT-PCR) and then conducted sequence and phylogenetic analyzes. In pathogenicity analysis, both high intra-cerebral pathogenicity index (ICPI) and mean death time (MDT) tests of chicken embryo were carried out. Furthermore, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with a LaSota vaccine strain were challenged by the recent Shaanxi strain was also performed.ResultsNine Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken flocks in China were genotypically and pathotypically characterized. Amino acid sequence analysis revealed that all the recent Shaanxi-isolated NDVs have (112)R-R-Q-K-R-F(117) for the C-terminus of the F2 protein and exhibit high ICPI and MDT of chicken embryos, suggesting that they were all classified as velogenic type of NDVs. Phylogenetic analysis of these isolates showed that they belong to subgenotype VIId that have been implicated in the recent outbreaks in northwestern China. The percentage of amino acid sequence identity of F protein between recent Shaanxi stains and five vaccine strains was in the range of 81.9 %-88.1 %, while the percentage of amino acid sequence identity of HN protein between recent Shaanxi strains and vaccine strains was in the range of 87.4 %-91.2 %. Furthermore, a number of amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of these isolates were observed, which may lead to the change of antibody recognition and neutralization capacity. A cross-protection experiment indicated that specific-pathogen-free chickens vaccinated with a LaSota vaccine strain was not capable of providing full protection for the flocks that were challenged by the recent Shaanxi strain.ConclusionsTaken together, our findings reveal that recent Shannxi NDVstrains exhibit antigenic variations that could be responsible for recent outbreaks of NDVs in northwestern China.

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Newcastle disease virus in pakistan: genetic characterization and implication in molecular diagnosis.

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Newcastle disease virus in pakistan: genetic characterization and implication in molecular diagnosis.

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