Background
Materials and methods
Ethics statement
Sample preparation
Viral metagenomic analysis
APPV confirmation by NS3 gene RT–PCR and sequencing
APPV genetic evolutionary analysis
Recombination, amino acid sequence, glycosylation analysis and antigen prediction
Results
Viral metagenomic analysis
Sequencing samples | Alignment subjects | Reads counts | Assembled sequences | |
---|---|---|---|---|
RNA | Atypical porcine pestivirus | 48 | Complete genome | |
Getah virus | 28 | Partial sequence | ||
Porcine kobuvirus | 6 | Partial sequence | ||
Hubei picorna-like virus 61 | / | Partial sequence | ||
Porcine picobirnavirus | / | Partial sequence | ||
Bovine picobirnavirus | / | Partial sequence | ||
Human picobirnavirus | / | Partial sequence | ||
Simian picobirnavirus | / | Partial sequence | ||
Posavirus 3 | / | Partial sequence | ||
Posavirus 1 | / | Partial sequence | ||
Porcine sapovirus | / | Partial sequence | ||
Mamastrovirus 3 | / | Partial sequence | ||
DNA | Po-Circo-like virus | / | Partial sequence | |
uncultured human fecal virus | / | Partial sequence | ||
Porcine serum-associated circular virus | / | Partial sequence | ||
Porcine polyomavirus | / | Partial sequence | ||
IAS virus | / | Partial sequence | ||
Porcine bocavirus 1 | / | Partial sequence | ||
Porcine parvovirus 1 | / | Partial sequence | ||
Porcine parvovirus 5 | / | Partial sequence | ||
Bovine herpesvirus type 1.2 | / | Partial sequence | ||
Bovine alphaherpesvirus 1 | 7 | Partial sequence | ||
Porcine circovirus 3 | / | Partial sequence |
APPV confirmation by NS3 gene RT–PCR and sequencing
Genome sequence and homology analysis of APPV
Regions | Clade I | Clade II | Clade III | |||
---|---|---|---|---|---|---|
nt | aa | nt | aa | nt | aa | |
polyprotein | 82.6–84.2 | 91.4–92.4 | 93.2–93.6 | 96.4–97.7 | 80.7–85 | 90.6–92.2 |
Npro | 78-81.5 | 79.4–83.3 | 90.9–92.6 | 90.6–93.9 | 77.4–77.8 | 80-81.1 |
C | 78.4–84.1 | 83.8–90.1 | 93.4–95.2 | 95.5–98.2 | 80.5–81.4 | 91–91 |
Erns | 80.8–83 | 89.5–94.3 | 91.9–94.4 | 95.7–98.1 | 80-80.6 | 92.4–93.3 |
E1 | 83.2–85.8 | 94-97.5 | 90.2–94 | 95.5–97.5 | 80.1–80.7 | 92.5–94.5 |
E2 | 84.1–86.9 | 91.3–95.9 | 92.1–93.6 | 94.2–96.7 | 80.6–81.2 | 89.6–92.1 |
P7 | 78.1–95.3 | 78.1–95.8 | 89.1–93.8 | 89.6–93.8 | 76.6–77.6 | 77.1–78.1 |
NS2 | 81.3–89.8 | 90.1–95.2 | 91.8–93.8 | 95.2–96.5 | 79.7–80.7 | 88.9–89.8 |
NS3 | 84.5–86 | 95.5–97.2 | 93.4–94.3 | 98.4–99.9 | 83.6–95 | 96.1–96.7 |
NS4A | 78.6–84.1 | 89.6–95.5 | 91–94 | 97-98.5 | 78.1–89.1 | 89.6–91 |
NS4B | 83.2–85.5 | 95-97.1 | 92.9–93.7 | 98.2–99.4 | 80.1–81.1 | 94.4–95.3 |
NS5A | 79.1–80.6 | 84.4–87.1 | 92.4–93.3 | 95.6–96.8 | 77.6–78.3 | 83.1–83.5 |
NS5B | 82.7–84 | 90.2–92.2 | 93.1–93.9 | 96.5–97.6 | 80.9–90.4 | 89.6–93.6 |
Phylogenetic analysis
Identity /Divergence | 1.2 | 1.4 | 1.5 | 1.6 | 1.7 | 2.1 | 2.2 | 2.3 | 3 |
---|---|---|---|---|---|---|---|---|---|
1.2 | 97.7 /2.3 | 89.1–89.3 | 88.7–89.3 | 87.6–88.1 | 85-85.3 | 79.2–80.9 | 81-81.1 | 80.4–80.6 | 79.7–80.2 |
1.4 | 10.7–10.9 | 98.4–99 /1-1.6 | 88.9–90 | 88.1–88.3 | 84.9–85.6 | 79.3–80.6 | 80.6 | 80.4–80.6 | 79.1–79.9 |
1.5 | 10.7–11.3 | 10-11.1 | 97.5–99 /1-2.5 | 86.8–87.6 | 84.7–86.7 | 78.7–80.2 | 79.3–80.7 | 79.1–80 | 78.7–79.7 |
1.6 | 11.9–12.4 | 11.7–11.9 | 22.4–23.2 | 100 /0 | 84.7–85 | 79-79.7 | 79.3 | 80 | 78.7–79.4 |
1.7 | 14.7–15 | 14.4–15.1 | 23.3–25.3 | 15-15.3 | 97.8–100 /0-2.2 | 78.9–80.5 | 79.2–79.4 | 79.3–80.1 | 77.1–78.7 |
2.1 | 19.1–20.8 | 19.4–21.3 | 19.8–21.3 | 20.3–21 | 19.5–21.1 | 94.6–99.9 /0.1–5.4 | 93.6–94.5 | 92.4–93.3 | 76.8–77.9 |
2.2 | 18.9–19 | 19.4 | 19.3–20.7 | 20.7 | 20.6–20.8 | 5.5–6.4 | 100 /0 | 92.7 | 77.5–77.9 |
2.3 | 19.4–19.6 | 19.4–19.6 | 20-20.9 | 20 | 19.9–20.7 | 6.7–7.6 | 7.3 | 100 /0 | 77.6–78.3 |
3 | 19.8–20.3 | 20.1–20.9 | 20.3–21.3 | 20.6–21.3 | 21.3–22.9 | 22.1–23.2 | 22.1–22.5 | 21.7–22.4 | 98.2–99.8 /0.2–1.8 |
Recombination analysis
Amino acid sequence analysis
Glycosylation analysis
Antigen prediction
Representive isolates | Clades/ Subclades | Patterns of putative N-glycosylation sites | TOP 15 most likely B-cell epitops residues |
---|---|---|---|
APPV-China/SWU-KZ/2018 | 1.2 | N51 + N64 + N103 | 5R, 22E, 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 173R, 190K, 191N, 192D, 193Y |
GX01-2019 | 1.4 | N64 + N103 | 18K, 35R, 36E, 38Q, 39E, 53R, 70R, 74R, 76R, 173R, 179R, 190K, 191N, 192D, 193Y |
APPV_GD | 1.4 | N51 + N64 + N103 | 22E, 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 173R, 179R, 190K, 191N, 192D, 193Y |
JX-JM01-2018A01 | 1.5 | N51 + N64 + N103 + N141 | 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 173R, 179K, 180K, 190K, 191N, 192D, 193Y |
GX-GL67 | 1.5 | N51 + N64 + N103 | 22E, 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 142D, 152E, 173R, 179R, 190K, 191N |
GX02-2018 | 1.6 | N51 + N64 + N127 + N103 + N141 | 22E, 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 173R, 179R, 190K, 191N, 192D, 193Y |
GX04/2017 | 1.7 | N51 + N64 + N103 + N141 | 22E, 35R, 36E, 38R, 39E, 70R, 71R, 74R, 76R, 173R, 179R, 180K, 190K, 191N, 192D |
APPV/CNYJ/2018 | 2.1 | N51 + N64 + N103 + N127 | 24R, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 88D, 173R, 179R, 180K, 190K, 191N, 193Y |
APPV-China/GD-SHT/2016 | 2.1 | N64 + N103 + N127 | 22E, 24R, 35R, 36E, 38R, 39E, 41R, 70R, 74R, 76R, 154E, 173R, 179R, 180K, 190K |
GX02-2019 | 2.1 | N51 + N127 | 5R, 35R, 36E, 38R, 39E, 62R, 70R, 74R, 76K, 88D, 173R, 183Y, 190K, 191N, 193Y |
GD-BH02-2018 | 2.1 | N51 + N64 + N127 + N103 + N141 | 35R, 36E, 38R, 39E, 70R, 74R, 139E, 173R, 179K, 180K, 181D, 183Y, 189K, 190K, 191N |
APPV-China/GD-SD/2016 | 2.2 | N51 + N64 + N103 | 18K, 35R, 36E, 38R, 39E, 70R, 74R, 76R, 173R, 179R, 180K, 190K, 191N, 192D, 193Y |
APPV-SDHY-2022 | 2.3 | N51 + N64 + N103 + N127 | 35R, 36D, 38R, 39E, 70R, 74R, 76R, 88D, 89N, 92E, 173R, 179R, 180K, 190K, 191N |
GD-HJ-2017.04 | 3 | N51 + N64 | 17E, 36E, 38Q, 39E, 70R, 76R, 139E, 173R, 179R, 180K, 189K, 190K, 191N, 192D, 193Y |
AH-SG-2018.01 | 3 | N64 | 18K, 36E, 38Q, 39E, 70R, 74K, 76R, 88D, 173R, 179R, 180K, 189K, 190K, 191N, 193Y |