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Abstract The aim of this work are: propagation and isolation of NzdV from supected samples collected from infected chickens using the embryonating chicken eggs biological and serological detection and identification of different loacl isolated on the basis of haemagglutination (HA) property and haemagglutination inhabition test using NDV specific antiserum.Newcastle disease (ND) is a highly contagious disease of birds and one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, several outbreaks have occurred in Egypt in the last 2 years and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of South Valley Provinces (Sohag, Qena and Luxor) during the period of 2011 - 2012.This study was carried out on 126 samples (liver, proventriculus, intestines, brain, spleen, lung, trachea and cecal tonsils), were collected from freshly dead and diseased live commercial broiler and backyard chicken flocks showing respiratory and/or nervous manifestations.Samples were prepared and inoculated into embryonating chicken eggs of 9 -11 days old via allantoic cavity for virus propagation. Inoculated eggs were observed for 5-7 days post-inoculation (PI) then the allantoic fluid was collected and tested for haemagglutination (HA) character by rapid and slow HA tests to confirm the virus growth. The positive samples for HA were identified by haemagglutination inhibition (HI) test using NDV specific antiserum to confirm the presence of the virus. The obtained results indicated that 45 (35.7 %) samples were NDV positive.Molecular confirmation of the virus was performed by one step reverse transcriptase polymerase chain reaction (RT-PCR) after the RNA extraction from the allantoic fluids of 45 ND positive samples. RT-PCR was conducted using specific primers to amplify 695 base pair (bp) fragment targeting region at the end of matrix (M) gene and Carboxyl terminus (C- terminus) of Fusion (F) gene, including cleavage site. RT-PCR results showed that all ND positive samples produced the specific PCR product at 695bp.The pathotyping of the isolated virus was carried out biologically by intracerebral pathogenicity index (ICPI) in 1-day-old chicks. The clinical manifestations and post mortem picture of NDV were observed on the inoculated chicks. The results revealed that The ICPI ranged from 1.4- 2 index which referred that all NDV isolates were ranged from moderate to velogenic typesIn order to understand the evolution of NDV in Upper Egypt and confirm the pathotyping, 8 out of 45 samples were selected from different regions for DNA sequencing and phylogenetic analysis. The post sequencing analysis of the cleavage site of the fusion gene showed that all the isolates have multiple basic amino acids at the C- terminus of the F2 protein and phenylalanine at residues 117 of the F1 protein, with cleavage site motif 112R-R-Q-K-R-F117, classifying them as velogenic which correlated to the results of ICPIThe obtained nucleotide sequences were aligned in compare with the LaSota and Egyptian strains previously isolated and the results indicated that all (8 strains) were 22 – 20% different from LaSota (vaccine strain) and 1-2 % from Egyptian one (2011), 16-22 % from other Egyptian one (2006, 2010). However, the 8 isolated were clustered into 6 clades based on nucleotide differences as well as away from the LaSota strain. The predicted amino acid sequences of these isolates were 21% different from Lasota also; some strains had a unique amino acid in different positionsThe nucleotide sequences were then compared with available sequences deposit in the Genebank and used to construct the phylogenetic tree. Phylogenetic analysis indicated that the current isolated strains belonged to genotype VII which is the predominant genotype responsible for most ND outbreaks since the end of the last century and it is currently circulating in Egypt, causing very high mortalities in chicken flocks. The obtained isolates were genetically distinct and phylogenetically distant from the LaSota strainEgyptian isolates (2006, 2010) and Israel (2003) while grouped with ND strains isolated from Egypt (2012) and Israel (2011). Far phylogenetic distance between vaccines and current obtained virulent strains which indicate to the occurrence of new ND stains different from the vaccine strains and other Egyptian strains isolated from chicken in 2006. These strains developed from the accumulation of point mutations in different sites of the F gene as well as this evolution may be a major reason for the continuous outbreaks of ND in Egypt.from the previous results we can conclude that: 1. Newcastle disease is still a major problem for the commercial and backyard poultry. 2. Presence of new NDV strains which differ from the vaccinal strains used for vaccination programs in Egypt so, studies on comparison of antigenic structure of the newly pathogenic NDV strains isolated from different outbreaks with that of the viruses used for the vaccination are of crucial importance to understand the causes of frequently occurring vaccine failure. 3. Poultry farmers need to be educated to adopt the biosecurity programs 4. A timely reporting mechanism should be developed that can be used to control an ND epidemic in the country |