Background
Methods
Ethics statement
Virus isolation
RNA extraction and PCR amplification
Sequencing and phylogenetic analysis
Prediction of glycosylation sites
Receptor-binding analysis
Antigenic analysis
Viral growth kinetics
Animal studies
Histology and immunohistology of mouse lung sections
Results
Virus isolation
Phylogenetic analysis of the ZJ-1664 and ZJ-1667 H6N1 strains
Segment | Position | Virus with the highest percentage of nucleotide identity | Genbank accession number | Homology (%) | Lineage |
---|---|---|---|---|---|
PB2 | 1–2280 | A/duck/Mongolia/520/2015(H1N1) | LC121393 | 99 | group III |
PB1 | 1–2274 | A/duck/Mongolia/66/2015(H10N2) | LC108118 | 98 | group III |
PA | 1–2151 | A/duck/Mongolia/66/2015(H10N2) | LC108119 | 99 | group III |
HA | 1–1701 | A/duck/Hubei/ZYSYG15/2015(H6N2) | KY415608 | 98 | group III |
NP | 1–1497 | A/wild bird/Korea/SK14/2014(H1N1) | KX066872 | 99 | group III |
NA | 1–1410 | A/wild bird/Wuhan/WHHN16/2014(H1N1) | KU143337 | 99 | Eurasian |
M | 1–959 | A/duck/Mongolia/543/2015(H4N6) | LC121415 | 99 | Europe |
NS | 1–838 | A/duck/Mongolia/543/2015(H4N6) | LC121416 | 99 | Allele II |
Molecular characterization of the ZJ-1664 and ZJ-1667 H6N1 strains
H3 numbering system | N-linked glycosylation sites in HA | HA cleavage | Receptor-binding site | Left edge of receptor-binding site | Right edge of receptor-binding site | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
320–329
|
98
|
153
|
155
|
183
|
190
|
194
|
195
|
220–229
|
134–138
| ||
H6 position
|
339–344
|
107
|
166
|
168
|
197
|
204
|
208
|
209
|
234–243
|
146–150
| |
ZJ-1664(H6N1) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVTKA |
ZJ-1667(H6N1) | 6 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVTKA |
A/chicken/Zhejiang/727031/2014(H6N2) | 5 | PQIETR | Y | W |
V
| H | E | L | Y | RPAVNGQRGR | GVSSA |
A/duck/Zhejiang/727038/2014(H6N2) | 6 | PQIETR | Y | W | I | H | E | L | Y | RPAVNAQRGR | GVSSA |
A/chicken/Zhejiang/727001/2014(H6N6) | 5 | PQIETR | Y | W |
V
| H | E | L | Y | RPAVNGQRGR | GVSSA |
A/chicken/Zhejiang/727028/2014(H6N6) | 6 | PQIETR | Y | W | I | H | E | L | Y | KPAVNGQRGR | GVSSA |
A/chicken/Zhejiang/110102/2015(H6N6) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPVVNGQRGR | GVTSS |
A/duck/Hubei/ZYSYG15/2015(H6N2) | 6 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVTKA |
A/chicken/Hunan/S4191/2009(H6N2) | 5 | PQIETR | Y | W |
V
| H | E | L | Y | RPAVNGQRGR | GVSSA |
A/duck/Shantou/2195/2003(H6N2) | 6 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVSSA |
A/wild duck/Shantou/2853/2003(H6N2) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVTSS |
A/duck/Hunan/573/2002(H6N2) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVTKA |
A/duck/Eastern China/48/2010(H6N6) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVSSA |
A/duck/Jiangxi/5178/2007(H6N2) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVSSA |
A/duck/Zhejiang/S4204/2010(H6N6) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVSSA |
A/swine/Guangdong/K6/2010(H6N6) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPVVNGQRSR | GVTSS |
A/muscovyduck/Vietnam/LBM601/2014(H6N6) | 5 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GVSSA |
A/mallard/Ohio/664/2002(H6N6) | 5 | PQVKTR | Y | W | I | H | E | L | Y | RPPANGQRGR | GVSSA |
A/Teal/Hong Kong/W312/97(H6N1) | 6 | PQIETR | Y | W | I | H | E | L | Y | RPAVNGQRGR | GTTRS |
A/Taiwan/2/2013(H6N1) | 6 | PQIATR | Y | W |
V
| H |
V
| L | Y | RPAVNGQRSR | GVTNA |
Prediction of glycosylation sites in the ZJ-1664 and ZJ-1667 H6N1 strains
Receptor-binding analysis of the ZJ-1664 and ZJ-1667 H6N1 strains
Virus | HA titers with normal chicken RBCs | HA titers with α-2,3 specific-neuraminidase-treated chicken RBCs | HA titers with sheep RBCs | Receptor specificity |
---|---|---|---|---|
ZJ-1664(H6N1) | 256 | 32 | 256 | α2,3-SA and α2,6-SA binding |
ZJ-1667(H6N1) | 256 | 32 | 256 | α2,3-SA and α2,6-SA binding |
A/chicken/Zhejiang/329/2011(H9N2) | 256 | < 2 | 256 | α2,3-SA binding |
A/Puerto Rico/8/1934(H1N1) | 256 | 256 | < 2 | α2,6-SA binding |
Antigenic analysis of the ZJ-1664 and ZJ-1667 H6N1 strains
Polyclonal antisera titers to | Reassortant H6N1 virus | |
---|---|---|
ZJ-1664 | ZJ-1667 | |
ZJ-1664(H6N1) | 256 | 256 |
ZJ-1667(H6N1) | 256 | 256 |
A/chicken/Zhejiang/727031/2014(H6N2) | 16 | 16 |
A/duck/Zhejiang/727038/2014(H6N2) | 16 | 16 |
A/chicken/Zhejiang/727001/2014(H6N6) | 32 | 32 |
A/chicken/Zhejiang/727028/2014(H6N6) | 16 | 16 |
A/chicken/ Zhejiang/110102/2015(H6N6) | 16 | 16 |
A/chicken/Zhejiang/C1/2013(H9N2) | 2 | 2 |
A/goose/Zhejiang/112071/2014(H5N1) | 4 | 4 |
Viral growth kinetics
Pathogenicity in mice
Virus | No. of survivors/no. tested | HI titer (log2) | Virus replication in experimentally infected mice Virus titers in organs of mice (log10 EID50/ml) | |||
---|---|---|---|---|---|---|
Tissue | 3 day | 6 day | 9 day | |||
ZJ-1664 | 6/6 | 6.33 ± 0.52 | Lung | 2.67 ± 0.57 | 3.67 ± 0.57 | 2.33 ± 0.57 |
Brain | 0 | 0 | 0 | |||
Heart | 0 | 1.33 ± 0.57 | 0 | |||
Liver | 0 | 0 | 0 | |||
Kidney | 0 | 0 | 0 | |||
Spleen | 0 | 0 | 0 | |||
ZJ-1667 | 6/6 | 5.83 ± 0.75 | Lung | 2.33 ± 0.57 | 2.67 ± 0.57 | 1.33 ± 0.57 |
Brain | 0 | 0 | 0 | |||
Heart | 0 | 0 | 0 | |||
Liver | 0 | 0 | 0 | |||
Kidney | 0 | 0 | 0 | |||
Spleen | 0 | 0 | 0 |