الفهرس 
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Abstract
In the current study, well identified field isolate of HPAI H5N1 subtype was used. The isolated was designed as A/chicken/ Kalyobia /ch2.2.18/2011 (H5N1) and the complete nucleotide sequence of N1 gene was submitted to the gene bank data base and have the accession number JQ791546. This virus was used in the cloning procedures and generation of the DNA vaccine (pDEST40/N1) coding for the neuraminidase gene.
First the virus seed lot was characterized before use by propagation in SPF-ECE, and after embryo death ( usually within 18 h) the chorio-allanotic fluid was collected. The propagated virus gave HA titer equal to 10 Log 2 and HI titer equal to 8 Log 2 when test with anti-H5 antiserum. No detectable HI titer was noticed when tested against NDV antiserum indication its purity.
Molecular characterization was done using traditional RT-PCR using 2 pairs of primer sets amplifying 300 bp fragments of both the matrix gene of Influenza A virus and the cleavage site of the H5 gene of HPAI-H5N1 subtype. As expected the seed lot gave amplicons with the predicted size for both genes.
The cleavage site of the seed viruswas subjected to sequencing to study the sequence of the polybasic amino acid at the cleavage site. The sequence was found to be GE↓RRRKKR↓GLF. This polybasic amino acid sequence is characteristic to the HPAI.
The full length N1 gene was amplified and sequenced. Sequencing of the full length NA1 gene and the deduced amino acid sequence revealed that the gene is 1400 bp. Several N glycosylation sites (2 sites) and protein kinase C phosphorylation sites (10 sites) were found on analysis of the sequence.
The B-cell epitope mapping revealed the presence of 45 predicted B-cell epitope when the threshold was set at 0.51.
After this extensive characterization, the N1 gene was amplified from the seed virus in 2 steps to prepare it for cloning. First the full gene was amplified with the internal primers that co-amplify the promoter like sequence at both 3’ and 5’ ends of the gene , then this amplicon was used as a template for amplification of gene insert with modification at the 5’ end to introduce the CCGA to facilitate the directional cloning and the 3’ end was also modified to remove the stopping codon to prevent premature termination of transcription.
The insert was then cloned in the entry vector pENTER/SD/D/Topo cloning vector and transformed in E.coli Topo10 competent cells . After overnight culture, plasmid miniprep was done and electrophoresed on 1% agarose. The plasmid migrates at about 4.0 Kbp corresponding to the size of the entry clone plus the insert.
In order to confirm the successful cloning and the presence of the N1 insert within the entry vector, Q-PCR experiment that utilize a pair of primers and a dual labelled probe was used. All tested plasmid preparations from the grown colonies on in the LB-kanamycin plates gave a Ct ranges from 32.66 – 34.49 while non transformed E. coli grown on non-antibiotic plates gave no Ct.
Homologues recombination was then done between the entry clone and the destination vector which will serve as the mammalian expression DNA vaccine (pDEST40). After transformation of E.coli Topo10 with the products of the recombination, colonies appear which subjected to miniprep. Plasmid electrophoresis showed product with 8.4Kbp representing the recombinant pDEST40/N1mammalian expression DNA vaccine.The successful cloning and the presence of the N1 insert within the pDEST 40 vector was confirmed by Q-PCR experiment, all the test plasmid preparations gave a positive Ct ranges from 14.19 – 15.91.
The pDEST 40/N1 DNA vaccine preparation was tested in comparison with the oil adjuvenated inactivated whole reassorted H5N1 virus vaccine commercially available. After challenge with 5.5 Log 10 EID50 of the HPAI virus, protection rate showed that the pDEST 40/N1 DNA vaccine failed to give the acceptable limit of protection which is the 80% according to the evaluation protocols of the central laboratory for evaluation of vet. Biologics which is the sole organization in Egypt allowed for evaluation of the veterinary vaccines. The DNA vaccine gave only 60% of protection compared to the inactivated vaccine which gave 80% protection.
On the other hand, shedding of the virus was surprisingly very limited with the pDEST 40/N1 DNA vaccine as it reduced virus shedding to 0.46 Log10 EID50 when compared to the reassorted inactivated H5N1 (the mean EID50 was 3.88 Log10). The control non vaccinated chickens gave a very high level of shedding (the mean EID50 was 5.24 Log10)
The live birds that survive the challenge also showed nearly no level of shedding for chicken vaccinated with the DNA vaccine while the those receiving the inactivated whole virus vaccine gave nearly the same results as the dead birds within the same group (mean EID50 1.74Log10).
As a conclusion, although the pDEST 40/N1 DNA vaccine gave lower rate of protection, yet it was superior in reducing virus shedding after challenge in dead birds and eliminated completely virus shedding in live birds survive the challenge.
As a recommendation, pDEST 40/N1 DNA vaccine could be co-administered with other vaccinal preparations to reduce shedding and prevent environmental load with the HPAI- H5N1 subtype.