الفهرس 
Introduction
Historical perspectiveOnly 14 pages are availabe for public view
 |  | from | 32 |  |  |
| | | from | 32 | | |
Abstract
Newcastle disease (ND) is a highly contagious viral disease and still remains number one disease affecting poultry industry in Egypt. It has attained an intricate state in the way that different isolates and strains of virus provoke tremendous variation in the severity of the outbreak.
Because of this variation, vaccination programmes launched against ND in the past could not reach complete accomplishment for its control. Extensive prophylaxis and control programmes against ND using different types of vaccines are in practice but absolute success in controlling the disease could not be accomplished.
It may be owing to the use of infective vaccines, antigenic, genotypic or phenotypic variations amongst different ND virus strains or the fallacious administration.
Thus, molecular epidemiology and phylogenetic analysis of NDV in Egypt, Africa and Middle East countries is an important tool to determine the current situation, trace the source of infection and to determine the possible root by which the virus spreads, assisting these countries in developing their disease control measures, which needs to be improved.
The destination from our study was the characterization of NDV isolated from outbreaks in the region of Upper Egypt provinces (Assiut, Sohag and Qena) during the period from 2012 to 2015 and determination of the phylogenetic relationship to the commercially available vaccinal strains.
116 tissue/organs samples (77 from Sohag, 23 from Assiut, 16 from Qena) were subjected to our study. Virus isolation was done by inoculation of embryonated chicken eggs (9-11) day old through the allantoic cavity by bacterial free suspension prepared previously from the homogenized samples.
The harvested allantoic fluid collected from dead embryos or at the end of (5-7) days post inoculation was tested by rapid and slow haemagglutination tests. The haemagglutination inhibition test was done by determination of 4HA unit of the HA positive samples and preparation of hyperimmune serum against NDV. Results revealed 100 sample (82.2%) were HI NDV positive.
Pathogenicity assessment in vivo was done by Intracerebral pathogenicity index (ICPI) in day old chick. The signs and lesions observed in inoculated chicks were indicating NDV infection. The value of ICPI ranged from (0.85- 1.6) indicating that our isolates were virulent (velogenic and mesogenic pathotypes).
For more confirmation of these results 46 isolates were subjected to molecular pathotyping. One step RT-PCR was done using Newcastle Disease virus-common and patho Detection kit. Forty four (44) isolates (95.65%) had product size of 204 bp expected Pathogenic NDV indicating they were NDV positive and virulent isolates.
Molecular characterization of the virus was done by performance of one step RT-PCR for the extracted viral RNA. PCR amplification and sequencing were performed using degenerative primers for obtaining 535 bp fragment length including fusion gene cleavage site. All samples subjected to one step RT-PCR gave the desired 535 PCR product. Gel extraction was done for further purification.
The purified PCR products of 12 isolates were selected for DNA sequencing and phylogenetic analysis.
Nucleotide and deduced amino acid sequence analysis of cleavage site of the F-gene of all field isolates revealed the motif 112 R-R-Q-R-R-F117 at the C-terminus of the F2 protein and F (phenylalanine) at the N-terminus of the F1 protein (residue 117) indicating that these strains were velogenic.
The nucleotide sequence analysis revealed that our isolates showed greatest nucleotide identities (99.3%) with the velogenic strain from Jordan (JQ176687-Chicken-Jordan-Jo11-2011-VIId (5d) and Israel Turkey strain ( JN979564-Turkey-Israel-111-2011-Israel VIId (5d) , proving that the virus circulating in Egypt is probably extending from Middle Eastern region.
The highest similarity with previously characterized Egyptian strains was (JX885868-chicken-VRLCU138-Egypt-2012) which was (98.6%). the homology between our isolates and genotype II representing strains was the least ranging from (83.9 % - 74.2 %)
Phylogenetic analysis showed that our isolates could be classified into three genotypes (VIId, VIIa and II) indicating that VIId is the predominating circulating genotype in Egypt (representing fourth panzootic) where 10 isolates (83.3%) were clustered. One isolate for genotype VIIa and one for genotype II.
Cluster of strain NDV-chicken-Egypt-Asy-11-2015 with genotype II could be explained that NDV strains responsible for ND sporadic outbreaks in vaccinated chickens can escape the immune responses, and thus contribute to the emergence of new genotypes.
Low evolution rate with Ka /Ks ratio ranged from (0.01 – 0.02) which is a value less than one indicating negative or purifying selection. The minimum evolutionary distance detected was (0.09) to genotype VIId , where the maximum distance was (0.21) to genotype II from which most commercially live virus vaccinal strains are derived. Thus, the control of NDV by vaccination still faces new challenges in different avian species.