CC BY
0mura et al., 2013).
RESULTS
Hemagglutination activity
After three blind passages only S3, S4, S7, S9, and S10 samples were positive for hemagglutination activity. S7 and S10 were positive for HA after the 1st egg passage, S3, and S4 were positive for HA after the 2nd egg passage, and S9 was positive for HA after the 3rd egg passage. RNA from 5 positive HA samples were sent for one-step RT-qPCR.
NDV detection by RT-qPCR
Only S4 was positive for Avian avulavirus 1 by RT-qPCR with a threshold cycle (CT) of 29.34 with a starting quantity of 3.033 log10 in comparison with a standard curve (Figure 2) with melting peak at 79 °C (Figure 3 and 4).
Amplification of full F and full HN proteins genes by RT-PCR
RT PCR products gel electrophoresis revealed the expected and correct size bands for full-length F and HN proteins genes.
Genetic and phylogenetic analysis
Fprotein gene
Blasting of sequence results obtained for the full F protein gene showed similarities with Chinese genotype VII strains with similarities varying from 95.5 % to 97.28% and with many Egyptian isolates varying from 94.5% to 95.5 %. The phylogenetic tree of the full F
protein gene showed that S4 isolate is closely related to genotype VII subtype D (Figure 5).
HN protein gene
Blasting of sequence results obtained for the full HN protein gene showed similarities with Chinese genotype VII strains with similarities varying from 95.69 % to 98.72% and with some Egyptian isolates varying from 94.9% to 95.45 %The phylogenetic tree of the full HN protein gene showed that S4 isolate is closely related to the Chinese genotype VII (Figure 6). Three-dimensional structure of F and HN monomer for S4 isolate was created by SWISS-Model modeling online server and visualized by PyMOL program version 2.3.4 (Figure 7 and 8).
1 2 3 4 5 6 7 i B !
50,0 C 95 DC 95 0 C \ 52 0C ! 60.0C G 0 T 0 3 95.0 C 550 C F
I 1000 / 005 > I j ft 10 \ £5.0 C /
/ 0-30 0:05 m N
jj 30:00 0:10 y [
r1 ! ! |4Dx
Figure 1. Thermal conditions applied at RT-qPCR and for the melting curve.
Amplification
0 10 20 30 40
Cycles
Figure 2. Threshold cycles of tested samples, green lines represent positive control samples (standard curve samples), the red line represents positive for Avian avulavirus sample (S4) appeared after 29.34 CT, and pink lines represent the negative for Avian avulavirus samples.
Figure 3. Melting curve of tested samples, green lines represent positive control samples (standard curve samples), the red line represents positive for Avian avulavirus sample (S4), and pink lines represent the negative for Avian avulavirus samples.
Figure 4. Melting peak of tested samples, green lines represent positive control samples (standard curve samples), the red line represents positive for Avian avulavirus sample (S4) showed a different melting peak, and pink lines represent the negative for Avian avulavirus samples.
i-(38) gi 705927774 < (45) gi 705927819 gt -(40) gi 705927791 i
b KM283610.1 NDV s KM288620.1 NDV str b KM288G 13.1 NDV s"
-(79) KC542909.1 NDV is
148 H77) KC542906.1 NDV is o >; J~1(80) KC542911.1 NDV isola L(82) KC542914.1 NDV isolâti
n NDV-B23-RLQP-CH-EG-12 NDV- B161 - RLQP- C H- E G-12 n NDV-B102-RLQP-CH-EG-12
-(44) gi 705927815 gb KM283619.1 NDV strain NDV-B127-RLQP-CH-EG-12
(43) gi 705927811 gb KM283G18.1 NDV strain NDV-B114-RLQP-CH-EG-12
-(37) gi 705927770 gb KM288609.1 NDV strain NDV-B7-RLQP-CH-EG-12
j—(41) gi 705927802 gb KM283616.1 NDV strain NDV-B110-RLQP-CH-EG-12
-(49) gi 823075337 gb KP316015.1 NDV isolate NDV-F460-RLQP-CH-EG-13
—(39) gi 705927787 gb KM288612.1 NDV strain NDV-B83-RLQP-CH-EG-12 (42) gi 705927807 gb KM288617.1 NDV strain NDV-B113-RLQP-CH-EG-12 —(50) gi 323075339 gb KP316016.1 NDV isolate NDV-F388-RLQP-CH-EG-14 -(56) gi 90684B883 dbj AB871428.1 NDV F gene for fusion protein strain: NDV EG CK67 11 —(53) gi 906848873 dbj AB871423.1 NDV F gene for fusion protein strain: NDV EG CK129 12 -(47) gi 014944470 gb KR010946.1 NDV strain VNDVVII chicken Egypt BSU-BS-KN2 2013 -(54) gi 906848879 dbj AB871426.1 NDV F gene for fusion protein strain: NDV EG CK 13 11 -(48) gi 814944476 gb KR010949.1 NDV strain VNDVVII chicken Egypt BSU-BS-KBB3 2014 -(55) gi 906846881 dbj AB871427.1 NDV F gene for fusion protein strain: NDV EG CK 101 12 (36) gi 4111G9SGS gb JX647839.1 NDV strain NDV-EG-5G7F-2012 (51) gi 891481119 gb KP119141.1 NDV strain EG CK NDV 44 Aswan 2013
-(46) gi 728054734 gb KM659400.1 NDV isolate chicken USC Egypt 2014
■P1) KC542912.1 NDV isolate Chicken China Shandong 01 2012 ■(24) FJ882014.1 NDV SG Liaoning2009 (VIId) (52) gi 891481134 gb KP119148.1 NDV strain EG CK NDV7B Qena 2012 (76) KC542905.1 NDV isolate Chicken China Liaoning 01 2009
□late Chicken China Shandong 02 2011 ate Chicken China Hebei 01 2010 :e Chicken China Beijing 01 2012 Chicken China Hebei 01 2012 —(78) KC542908.1 NDV isolate Chicken China Shandong 01 2011 —(¡35) KP742770.1 NDV isolate Sheldrake duck China Guizhou SS1 2014 (71) KC542899.1 NDV isolate Chicken China Jilin 01 2000 -(75) KC542903.1 NDV isolate Chicken China Beijing 02 2009 ¿57) gi 930308421 gb KR082481.1 NDV strain NDV House sparrow Desouk Egypt MS12 2014 i42~l(5S) gi 930306500 gb KR082482.1 NDV strain NDV House sparrow Qallin Egypt MS14 2014 ■(80) gi 930306822 gb KR082485.1 NDV strain NDV Cattle Egret Desouk Egypt MS3II 2014 -(16) DQ659677.1 NDV strain NA-1 (Vlld)
-(69) KC542897.1 NDV isolate Chicken China TianjinOI 2007
■(81) GQ849007.1 NDV isolate JSD0812 ■(74) KC542902.1 NDV isolate Chicken China Beijing 01 2009 —(67) JN986838.1 NDV strain APMV-1 chicken ZAAL495 04
1311(72) KC542900.1 NDV isolate Chicken China Liaoning 01 2008 Scrip's) KC542901.1 NDV isolate Chicken China Liaoning 03 2008
L(70) KC542898.1 NDV isolate Chicken China Tianjin 02 2007 —(22) EU533503.1 NDV isolate Hebei China 2004 lyild) —(88) KC542895.1 NDV isolate Chicken China Hebei 01 2006
PO) MK124761.1 Avian avulavirus 1 isolate goose/China/ZJ-1/2000 complete genome -(19) EF589134.1 NDV strain H2 (Vlld) 6 |(5) AF458010.1 NDV isolate JS-3 00 (Vile)
-(15) DQ439910.1 NDV strain NDV05-055 (VIIc) i13) DQ227246.1 NDV isolate JS02 (VIIc) (14) DQ227254.1 NDV isolate SWS03 (VIIc) 1 NDV strain Q-GB 445 97 (Vila) >) AF0839G1.1 NDV strain TW 94P (Vila) i) U82820.1 NDU62620 NDV Taiwan 95 (Vila) L(1) AF001134.1 NDV strain RI-1 88 (Vile)
■) KR732614.1 NDV strain NDV peacock Peru 2011 Y928933.1 NDV isolate MK13 75 (Vllb)
-(63) HQ589257.1 NDV strain Bareilly (Vllb) 2) GU182323.1 NDV strain chicken SPVC Karachi NDV 43 2008 (Vllf) j) JN682185.1 NDV isolate chicken CP Rawalpindil 2010 (Vllt) -(68) JN682189.1 NDV isolate chicken CP Rawalpindi2 2010 (Vllf) |(7) AY562986.1 NDV isolate anhinga U.S.(FI) 44083 93
-(17) EF065682.1 NDV isolate rAnhinga 1 NDV isolate chicken U.S.(CA) 1
-(27) gi 285020449 gb FJ969394.1 NDV strain NDV chicken Egypt 3 2006
'rC
Egypt VRLCU 2D14
Egypt2008
NDV chicken Egypt 4 2006
NDV II VRLCU Gharbia19 2009 -(30) gi 397870241 gb HQ455806.2 NDV isolate NDV II VRLCU BehairaO 2009 —(34) gi 397870249 gb HQ455810 2 NDV isolate NDV II VRLCU Giza23 2009
-(29) gi 397870239 gb HQ455805.2 NDV isolate NDV II VRLCU Giza27 2009
-(32) gi 397870245 gb HQ455808.2 NDV isolate NDV II VRLCU GharbialB 2009
(35) gi 397B70251 gb HQ455811.2 NDV Isolate NDV II VRLCU Gl7a24 2009 -(31) gi 397870243 gb HG455807 2 NDV isolate NDV II VRLCU Gbarbia17 2009 (84) JF950510.1 NDV strain LaSota
Figure 5. Neighbor-joining phylogenetic tree of the full-length F gene of Egyptian isolate of Newcastle disease virus (NDV) (S4) in comparison to other NDV strains from GenBank. Bootstrap values are shown above the branches. S4 isolate is indicated by a solid green circle.
Figure 6. Neighbor-joining phylogenetic tree of the full-length HN gene of Egyptian isolate of Newcastle disease virus (NDV) (S4) in comparison to other NDV strains from GenBank. Bootstrap values are shown above the branches. S4 isolate is indicated by a solid green circle.
Figure 7. 3D structure for F protein of Newcastle disease virus (S4 isolate) created by SWISS-Model modeling online server and visualized by PyMOL program version 2.3.4, red color represents the cleavage site.
Figure 8. 3D structure for HN protein of Newcastle disease virus (S4 isolate) created by SWISS-Model modeling online server and visualized by PyMOL program version 2.3.4.
DISCUSSION
In the current study, only five samples (50% samples) showed HA positive activity indicating infection with a hemagglutinating virus. To confirm NDV infection, RT-qPCR was performed using the HA positive samples.
Only S4 isolate was positive for NDV using universal primers for APMV-1. Negative RT-qPCR results for S3, S7, S9, and S10 may indicate an infection with another hemagglutinating virus-like avian influenza H9 or H5; however, history of mortalities and symptoms severity indicated H9 infection mixed with other respiratory pathogens other than H5 (Hussein et al., 2014; Sedeik et al., 2018). The most important pathogenicity indicator for NDV is the F protein gene sequence analysis mainly for cleavage site in which velogenic strains have polybasic amino acid sequences; therefore, molecular identification and phylogenetic analysis of the F gene is a major determinant
of NDV virulence instead of conventional methods (Mohamed et al., 2011; Damena et al., 2016). Also, it can be considered as a reliable way for NDV virulence evaluation when compared to traditional ways of evaluation (Ganar et al., 2014).
Results of F protein gene sequencing revealed that the cleavage site motif of S4 isolate has the sequence of velogenic NDV strains n2RRQKRF117 in agreement with (Sedeik et al., 2019). Also, the neurological effects of NDV infections by is thought to be due to the presence of the phenylalanine (F) residue at position 117 (Collins et al., 1993). The full sequence of both F and HN protein genes were submitted to the GenBank database with accession number MN905162 for the full F protein gene sequence and MN905163 for the full HN protein gene sequence.
F and HN proteins genes genetic and phylogenetic analysis in the present study revealed high similarity of S4 isolate with Chinese isolates and relatively fewer similarities with the Egyptian isolates which may strongly refer to the role of migratory wild birds in NDV evolution in Egypt.
CONCLUSION
Newcastle disease still occurs in sporadic cases despite massive vaccination programs implemented in the Egyptian poultry field. Migratory wild birds are supposed to have a big role in the continuous evolution of NDV in Egypt. Further epidemiological and surveillance work is strongly recommended to define the exact role of
migratory wild birds in NDV evolution in Egypt with defining the main causes of the inability of currently used vaccines to protect chickens against infection with Newcastle disease virus.
DECLARATION
Authors' contributions
All authors reviewed the final manuscript. This work is a part of Mira Maher, and Abdulrahman S. Metwally thesis under the supervision of Shakal M, and Gehan Safwat. Shakal M. designed, supervised the experiments, and co-wrote the paper. Gehan Safwat co-designed the experiment and co-wrote the paper. Mohammed A. Abdel Sabour conducted samples pooling, virus isolation, and co-wrote the paper. Mira Maher and Abdulrahman S. Metwally conducted RNA extraction, genes amplification by PCR, and conducted genetic alignment. Yahia M. Madbouly conducted RNA extraction, real-time reverse transcription PCR, GenBank submission, phylogenetic analysis, and co-wrote the paper.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Authors gratefully acknowledge Dr. Mohammed Rohaim, Virology Department, Cairo University, Egypt for his kind support and comments.
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