| Newcastle disease (ND), which is caused by Newcastle disease virus, is a member of Rubulavirus, Paramyxoviridae and is the highly contagious viral disease. Waterfowls were considered to be the natural reservoir of NDV. Duck were usually considered to be resistant to virulent NDV strains and mostly harbor lentogenic strains. However, ND from the duck constantly occured since1999.Seven isolates of virus were isolated from live poultry market of Guangxi in the year2008and2010, which were able to agglutinate chicken red blood cells. These isolates were neutralized by Newcastle disease virus standard antisera and were not neutralized by avian influenza virus (AIV) various sutypes standard antisera. They were confirmed to be Newcastle disease virus based on the results of HA test, HI test and RT-PCR.7isolates gave the mean death time (MDT) in embryonated chicken eggs ranging from93.6h-162h and ELD50between10-6.786/0.1mL-10-8.438/0.1mL,3isolates gave intracerebral pathogenicity index (ICPI) in day-old chickens between0.15-0.30, which demonstrated that7isolates were avirulent Newcastle disease virus.For the purpose to understand the molecule epidemiology characteristics of seven NDV strains from duck, including GX16/2008, GX17/2009, GX18/2009, GX19/2009, GX20/2010, GX21/2010and GX22/2010. Complete nucleotide sequences of these strains were acquired by reverse transcription-polymerase reaction. The entire genomic sequences of seven NDV strains consisted of1586 nt, which was the same in length to LaSota, Bl, Clone30, Ireland Ulster67strains. Deduced amino acid sequence of the cleavage site of fusion (F) protein revealed that all isolates had avirulent motifs. Of the7isolates,5exhibited sequence motif of112G-K-Q-G-R-L117at the cleavage site,2exhibited112G-R-Q-G-R-L117. Phylogenetic analysis based on comparison with different NDV sraines revealed that5isolates were phylogenetically close to genotype I NDVs while2were close to genotype II.The results of identity analysis of nucleotide and amido acid sequences showed that the identities of these strains between HM125898WDK/JX, were higher than of F48E8, Mukteswar strains.A set of primers were designed according to the F fusion gene sequences of NDV. The reaction temperature and the concentration of Betaine and Mg2+were optimized to develop a loop-mediated isothermal amplification assay for detection of NDV. Distinguishing different virulent strains of NDV and the attenuated strains were successfully detected by the LAMP assay. As a result of the sensitivity and specificity of the assay with a sensitive, simple, rapid and practical method for the detection of NDV in the field.Two sets of specific primers were designed according to the sequences (NDV and DPV in GenBank. It was shown that all samples containg NDV and DP could be amplified into two specific bands,419bp for NDV and602bp for DPV b this duplex PCR, but no specific band was amplified from other avian pathogen virus and bacteria. As little as0.4pg of NDV RNA and126pg of DPV DNA cou be detected by this duplex PCR. |
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