virus from naturally infected chickens in northern Iraq kurdistan Region Salahaddin University Erbil Agriculture Faculty Animal Science Department Authors Prof Dr Shony ID: 1042938
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1. Molecular identification and Genotyping of new high virulence Newcastle Disease virus from naturally infected chickens in northern Iraq (kurdistan Region)Salahaddin University - Erbil Agriculture FacultyAnimal Science DepartmentAuthors:Prof. Dr. Shony M. Odisho / Baghdad UniversityAssist. Prof. Dr. Rebah N. Al-Gafari /Nahren University Dr. Ahmad Ibrahim Ahmed / Salahaddin University-Erbil
2. IntroductionNewcastle Disease (ND) is an acute infectious viral disease , its wide range host and worldwide distribution in domestic poultry: (Mortality is 100% in chickens and 25-50% in vaccinated chickens ND virus were circulated in Iraq Since 1968 also isolated in 1979, 2005.Velogenic NDv strains is spread the worldwide, lastly isolated in Iraq by Ahmed et al 2018 1
3. IntroductionGenus Avulavirus in family ParamyxoviridaeThe genome is SS-SRNA consisting of 15186 ntThe genome contains six genes in the order of 3’-NP-P-M-F-HN-L-5’There are F and HN proteins form external envelope spikes 2
4. The aims of the current study were the molecular identification, genotyping and phylogenetic analyses of the new high virulence circulating NDV in naturally infected chicken from northern Iraq, Kurdistan region. The aim of the study4
5. 2. Propagation, Isolation and Identification Propagation and Isolation via several passages of ECE inoculationIdentification via: HA , HI with SHIS* Specific hyperimmune serum (SHIS) prepared according to (Iqbal, 2006), the most frequent titer serum selected and preserved at -20°C until further use. 14LocationSampleType of chickenAge (day)No. passage in ECE(log2) HA(log2) HIErbilE1Broiler28312864E2Broiler 32312864SulymaniyahS1Broiler 3633216S2Broiler 3033216DuhokD1Broiler 353512256D2Broiler 28312864
6. The genomic viral RNA was extracted from allantoic fluid (RNA extraction kit Patho Gene-spinTM) RNA was used as a template for RT-PCR amplification. Specific primers designed two partial of F gene:The optimal condition (Gradient PCR) and , cDNA template (1.5-2μl) were used
7. Six extracted viral RNA were used as a source for amplification following the steps to generate cDNA using Accu Power® Rocket Script TM RT PreMix (Bioneer from Republic of Korea) following the manufacture protocol and instructions, RT-PCR reaction was set up in a total volume of 40 μl per reaction Multiple alignments were done by Clustalx2 (15). Subsequent phylogenetic analysis was performed using MEGA6 program freely available
8. Results
9. Name of SampleType of substitutionpercentageRange of nucleotideSequence IDScoreExpectedIdentitiesSourceE1Transition3/7(42.9%)5115 to 5469KX496964.16093e-17098%U62620 TW/95 genotype VII NDVTransversion4/7(57.1%)S1Transition3/17(17.6%)719 to 856KF113350.11682e-3887%U62620 TW/95 genotype VII NDVTransversion14/17(82.4%)S2Transition3/11(27.3%)661 to 966KF113349.14931e-13596%U62620 TW/95 genotype VII NDVTransversion8/11(72.7%)Table 1: Transitions and transversion of nucleotide change in percentage of F1 gene.
10. 48Local NDVs Figure 1 Phylogenetic tree of local NDVs( red) using F1 gene
11. Name of SampleTypeof substitutionpercentageRange of nucleotideSequence IDScoreExpectedIdentitiessourceE1Transition6/19 (66.7%)5719 to 5865KT355595.11795e-4187%NDV isolate IBS025/13Transversion13/19(33.3%)E2Transition9/29 (31.1%)1065 to 1298AF164966.12919e-7588%NDV(OW/TW/2209/95Transversion20/29 (68.9%)D2Transition4/11 (36.4%)1074 to 1298AF164966.13563e-9495%NDV(OW/TW/2209/95Transversion7/11 (63.6%)S1Transition0/2 (0.0%)885 to 1317KY549653.17730.095%NDV(OW/TW/2209/95Transversion2/2(100%)S2Transition8/31 (25.8%)1111 to 1298AF164966.11873e-4383%NDV(OW/TW/2209/95Transversion23/31(74.2%)Table(2): Transitions and transversion of nucleotide change in percentage of F2 gene.
12. 49Local NDVs Figure 2 Phylogenetic tree of local NDVs( red) using F1 gene
13. Table 3 RT-PCR results of amplification of F2 and M gene isolated from local NDV particles (Borehingher Institut für Virusdiagnostic)Origin .No.NR.No.Types of SampleM.gene PCRF.gene LentogenicF.gene PathogenicFgene (2.VII)D1IQ|NRo56|2017AAfnegnegnegnegD2IQ|NRo57|2017AAf++++neg+++++E1IQ|NRo58|2017AAf+++neg++++E2IQ|NRo59|2017AAf+++neg++++S1IQ|NRo60|2017AAf++negneg++S2IQ|NRo61|2017AAf++neg(+)++AAF= Avian Allantoic Fluid, Threshold cycle TC: ++++=TC<19, +++ =TC20-24, ++ =TC25-29, + =TC30-35, (+) =TC36-39, neg=negative, E ,E2 are Erbil isolates , S1,S2 are Sulaimanihya isolates and D1,D2 are Duhok Isolates
14. Local NDVsFigure 3 Phylogenetic analysis of NDV positive diagnostic samples Analysis is based on first 436 nucleotides from coding Fusion protein gene (F2-fragment), applying Neighbor joining method, with test of phylogeny by bootstrap method with 1000 replications (A). Radial view.
15. Local NDVsFigure 4 Phylogenetic analysis of NDV positive diagnostic samples. Analysis is based on first 436 nucleotides from coding Fusion protein gene (F2-fragment), applying Neighbor joining method, with test of phylogeny by bootstrap method with 1000 replications; depicting that samples NR58-61/17 can be grouped as velogenic NDV belonging to genotype class II genotype VIIi.
16. Identification NDV in vaccinated broiler flocks from Northern Iraq was velogenic NDV which is still an on-going threat to poultry flocks. Genotype of isolated gene were showed type VIIi, which suggested in the production of an effective vaccine or choice the matched vaccine against this infection which reduced the economic lost dramatically. The presence of Iraqi/NDV/Vlli strains in the Northern of Iraq (NDV isolates registered as GenBank: MF678802.1.) lead to complicated and increased the cost of attempts control program.Conclusion
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