Serological and Molecular Characterization of Avian Metapneumovirus in Chickens in Northern Vietnam
Abstract
:1. Introduction
2. Materials and Methods
2.1. Farm Selection, Sample Collection, and Processing
2.2. Serology
2.3. Detection of aMPV by RT-Nested PCR
2.4. Sequencing of the Attachment Glycoprotein Gene (G Gene) and Genetic Analyses
3. Results
3.1. Seropositivity and Virus Positivity Rates of aMPV in the Northern Provinces of Vietnam
3.2. Seropositivity and Virus Positivity Rates of aMPV According to Age-Group
3.3. Genetic Characterization of the Circulating Strains of aMPV
Nucleotide position | 20 | 22 | 57 | 91 | 102 | 138 | 142 | 176 | 200 | 201 | 214 | 241 | 251 | 254 | 306 | 314 | 333 | 356 | 366 | 430 | 443 | 449 | 451 | 474 | 485 | 500 | 505 |
Nemovac_MZ574139 | T | A | G | A | A | T | T | G | C | G | T | A | G | G | G | G | A | G | T | G | C | A | G | T | G | A | C |
Hipraviar_MZ574138 | T | A | G | A | A | T | T | G | C | G | T | A | G | G | G | G | A | G | T | G | C | A | G | T | G | A | C |
GT1906_MZ574140 | ? | ? | ? | A | A | T | T | G | C | G | T | A | G | G | G | G | T | G | C | ? | ? | ? | ? | ? | ? | ? | ? |
GK9147_MZ574141 | ? | ? | ? | A | A | T | T | G | C | G | T | A | G | G | G | G | T | G | C | ? | ? | ? | ? | ? | ? | ? | ? |
GA2007_MZ574142 | ? | ? | ? | A | A | T | T | G | C | G | T | A | G | G | G | G | T | G | C | ? | ? | ? | ? | ? | ? | ? | ? |
GA1905_MZ574144 | ? | ? | ? | A | A | T | T | G | C | G | T | A | G | G | G | G | T | G | C | ? | ? | ? | ? | ? | ? | ? | ? |
GE20117_MZ574143 | ? | ? | ? | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | ? | ? | ? | ? | ? | ? | ? | ? |
Ck.1_MZ393666 | T | A | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.6_MZ393677 | T | A | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.2_MZ393668 | T | A | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.4_MZ393673 | T | A | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.3_MZ393671 | T | A | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.7_MZ393679 | ? | ? | G | G | A | T | A | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.8_MZ393681 | ? | ? | G | G | A | T | T | G | C | A | T | A | G | A | G | G | T | G | T | G | C | A | G | T | G | A | C |
Ck.9_MZ393682 | C | G | G | G | A | T | T | G | T | G | T | A | G | A | A | G | T | A | T | A | A | A | G | T | G | A | A |
Ck.5_MZ393674 | C | A | A | G | G | C | T | A | T | G | C | G | A | A | G | A | T | A | T | A | C | G | A | T | A | G | A |
Nucleotide position | 510 | 514 | 516 | 525 | 545 | 549 | 568 | 572 | 576 | 577 | 580 | 585 | 600 | 603 | 618 | 619 | 627 | 644 | 645 | 654 | 656 | 661 | 687 | 690 | 699 | 708 | 718 |
Nemovac_MZ574139 | A | A | G | T | C | T | C | T | T | T | A | G | T | T | G | C | C | G | C | T | T | A | T | A | C | T | A |
Hipraviar_MZ574138 | A | A | G | T | C | T | C | T | T | T | A | G | T | T | G | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.1_MZ393666 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.6_MZ393677 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.2_MZ393668 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.4_MZ393673 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.3_MZ393671 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.7_MZ393679 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | T |
Ck.8_MZ393681 | C | A | G | T | C | T | C | T | T | T | G | G | T | T | A | C | C | G | C | T | T | A | T | A | C | T | A |
Ck.9_MZ393682 | A | A | A | C | T | C | C | C | C | C | G | A | C | C | G | C | A | A | T | C | C | G | C | G | C | C | A |
Ck.5_MZ393674 | A | G | G | T | C | C | T | T | T | T | G | G | T | C | A | A | A | G | C | T | T | G | T | G | C | C | A |
Nucleotide position | 719 | 730 | 736 | 742 | 745 | 746 | 753 | 756 | 772 | 793 | 805 | 808 | 810 | 821 | 826 | 832 | 837 | 840 | 845 | 846 | 870 | 873 | 881 | 892 | 893 | 896 | 900 |
Nemovac_MZ574139 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | A | A | A | A | C | C | C | T | C |
Hipraviar_MZ574138 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | A | A | A | A | C | C | C | T | C |
Ck.1_MZ393666 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | G | A | A | A | C | C | C | T | C |
Ck.6_MZ393677 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | A | T | G | A | A | A | C | C | A | T | C |
Ck.2_MZ393668 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | G | A | A | A | C | C | C | T | C |
Ck.4_MZ393673 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | G | A | A | A | C | C | C | T | C |
Ck.3_MZ393671 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | G | A | A | A | C | C | C | T | T |
Ck.7_MZ393679 | T | C | G | T | G | G | A | A | C | A | A | A | G | G | C | G | C | T | G | A | A | A | C | C | C | T | C |
Ck.8_MZ393681 | T | C | G | T | G | G | A | A | T | A | A | A | G | G | C | G | C | T | G | A | A | A | T | T | C | T | C |
Ck.9_MZ393682 | A | A | A | C | A | G | A | G | C | A | G | A | G | A | C | A | C | C | A | G | A | A | C | C | C | C | C |
Ck.5_MZ393674 | T | A | G | T | A | A | C | G | C | G | G | G | A | A | T | G | C | C | A | A | G | G | C | C | C | T | C |
Nucleotide position | 914 | 915 | 926 | 938 | 961 | 972 | 975 | 977 | 979 | 980 | 985 | 1003 | 1010 | 1029 | 1032 | 1038 | 1068 | 1088 | 1132 | 1137 | 1150 | 1156 | 1187 | 1201 | 1212 | ||
Nemovac_MZ574139 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | G | G | ||
Hipraviar_MZ574138 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | ? | ? | ? | ? | ? | ? | ? | ? | ||
Ck.1_MZ393666 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | G | G | ||
Ck.6_MZ393677 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | G | G | ||
Ck.2_MZ393668 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | G | G | ||
Ck.4_MZ393673 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | G | G | ||
Ck.3_MZ393671 | C | A | C | T | A | A | T | C | A | T | A | A | C | T | T | T | A | C | C | T | T | G | G | G | G | ||
Ck.7_MZ393679 | C | A | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | ? | ? | ||
Ck.8_MZ393681 | C | G | C | T | A | A | T | C | A | T | A | A | T | T | T | T | A | C | C | T | T | G | G | ? | ? | ||
Ck.9_MZ393682 | A | A | T | C | A | G | C | T | G | C | G | G | T | C | C | C | G | T | T | T | T | A | C | A | G | ||
Ck.5_MZ393674 | C | A | T | C | G | A | C | C | A | T | A | A | T | T | T | T | A | C | T | C | C | A | G | G | A |
3.4. Phylogenetic Classification of Vietnamese aMPV Strains
4. Discussion
4.1. The Detection Rates of aMPV Infections
4.2. The Detection Rates of aMPV Infections According to Age-Group
4.3. The Genetic Characterization of Vietnamese aMPVs
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
PCR Type | Forward/Reverse | Sequences (5′-3′) | References |
---|---|---|---|
Outer | APV1/2.444G6- | CTGACAAATTGGTCCTGATT | [28] |
APV1.444G1+ | GGGACAAGTATCYMKAT | ||
Inner | APV1.268.361G5- | CAAAGARCCAATAAGCCCA | [28] |
APV1.268G8+ | CACTCACTGTTAGCGTCATA | ||
APV1.361G9+ | TAGTCCTCAAGCAAGTCCTC |
PCR Type | Forward/Reverse | Thermal Profile |
---|---|---|
Outer | APV1/2.444G6- | 1X (94 °C-4 min) 40X (94 °C-20 s, 50 °C-30 s, 72 °C-30 s) 1X (72 °C-5 min) |
APV1.444G1+ | ||
Inner | APV1.268.361G5- | 1X (94 °C-4 min) 40X (94 °C-20 s, 50 °C-40 s, 72 °C-60 s) 1X (72 °C-5 min) |
APV1.268G8+ | ||
APV1.361G9+ |
Forward/Reverse | Sequences (5′-3′) | References |
---|---|---|
Gstart+ | CAAGTATCCAGATGGGGTC | [29] |
/GB1- | GTACAGCACCACTCAATCAG | |
428.F.s2.G | GCTTATTGGCTCTTTGTGA | This study |
/428.R.s2.G | CGGAGTTGCTGTGCTGT |
PCR Type | Forward/Reverse | Thermal Profile |
---|---|---|
First PCR | Gstart+ /GB1- | 1X (94 °C-4 min) 40X (94 °C-20 s, 53 °C-30 s, 68 °C-60 s) 1X (72 °C-5 min) |
Second PCR-1 | 428.F.s2.G /GB1- | 1X (94 °C-4 min) 40X (94 °C-20 s, 53 °C-30 s, 68 °C-60 s) 1X (72 °C-5 min) |
Second PCR-2 | Gstart+ /428.R.s2.G | 1X (94 °C-4 min) 40X (94 °C-20 s, 53 °C-30 s, 68 °C-60 s) 1X (72 °C-5 min) |
Age * (Week) | aMPV Nested RT-PCR | aMPV ELISA | ||||
---|---|---|---|---|---|---|
No. Swabs/Organs | Positive | % Positive | No. of Serum | Positive | % Positive | |
3 | 24 | 5 | 20.8 | 15 | 4 | 26.7 |
4 | 19 | 2 | 10.5 | 26 | 11 | 42.3 |
5 | 3 | 1 | 33.3 | 9 | 0 | 0.0 |
6 | 23 | 6 | 26.1 | 79 | 6 | 7.6 |
7 | 12 | 1 | 8.3 | 5 | 0 | 0.0 |
8 | 10 | 3 | 30.0 | 10 | 1 | 10.0 |
9 | 11 | 2 | 18.2 | 18 | 13 | 72.2 |
10 | 13 | 4 | 30.8 | 15 | 15 | 100.0 |
11 | 16 | 2 | 12.5 | 26 | 14 | 53.8 |
12 | 21 | 2 | 9.5 | 60 | 16 | 26.7 |
13 | 6 | 1 | 16.7 | 5 | 4 | 80.0 |
14 | 20 | 9 | 45.0 | 19 | 16 | 84.2 |
15 | 9 | 1 | 11.1 | 9 | 7 | 77.8 |
16 | 9 | 0 | 0.0 | 2 | 2 | 100.0 |
17 | 1 | 0 | 0.0 | |||
18 | 6 | 0 | 0.0 | 6 | 4 | 66.7 |
19 | 1 | 0 | 0.0 | |||
20 | 3 | 0 | 0.0 | 2 | 0 | 0.0 |
21 | 1 | 0 | 0.0 | |||
24 | 2 | 0 | 0.0 | |||
26 | 1 | 0 | 0.0 | |||
28 | 3 | 0 | 0.0 | 11 | 0 | 0.0 |
32 | 1 | 0 | 0.0 | |||
37 | 1 | 0 | 0.0 | |||
48 | 1 | 0 | 0.0 | 10 | 10 | 100.0 |
60 | 1 | 0 | 0.0 | |||
96 | 1 | 0 | 0.0 | |||
NA ** | 37 | 5 | 13.5 | |||
Total | 256 | 44 | 17.2 | 327 | 123 | 37.6 |
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Nguyen, V.-G.; Chung, H.-C.; Do, H.-Q.; Nguyen, T.-T.; Cao, T.-B.-P.; Truong, H.-T.; Mai, T.-N.; Le, T.-T.; Nguyen, T.-H.; Le, T.-L.; et al. Serological and Molecular Characterization of Avian Metapneumovirus in Chickens in Northern Vietnam. Vet. Sci. 2021, 8, 206. https://doi.org/10.3390/vetsci8100206
Nguyen V-G, Chung H-C, Do H-Q, Nguyen T-T, Cao T-B-P, Truong H-T, Mai T-N, Le T-T, Nguyen T-H, Le T-L, et al. Serological and Molecular Characterization of Avian Metapneumovirus in Chickens in Northern Vietnam. Veterinary Sciences. 2021; 8(10):206. https://doi.org/10.3390/vetsci8100206
Chicago/Turabian StyleNguyen, Van-Giap, Hee-Chun Chung, Hai-Quynh Do, Thanh-Trung Nguyen, Thi-Bich-Phuong Cao, Ha-Thai Truong, Thi-Ngan Mai, Thi-Trinh Le, Thi-Hoa Nguyen, Thi-Luyen Le, and et al. 2021. "Serological and Molecular Characterization of Avian Metapneumovirus in Chickens in Northern Vietnam" Veterinary Sciences 8, no. 10: 206. https://doi.org/10.3390/vetsci8100206