The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Figures & data

Fig. 1 Impact of mutation at published mAb escape sites on haemagglutination inhibition titres performed with polyclonal chicken antisera.

Change in log₂ HI titre associated with stated amino-acid replacements, based on eight individual anti-UDL1/08 antisera with a minimum of five technical repeats, are indicated by cell colouring according to the colour legend with non-significant differences shown in grey and untested substitutions in white. Published mAb escape mutations are shown to the left of centre, whereas alternative substitutions are shown to the right. Mutations that introduce absent or almost absent amino-acid states ( < 1% of sequenced natural isolates) are labelled in smaller, italicised, grey font. * addition of potential glycosylation site. ^† would not rescue. ^‡ Q146H rescued with additional substitution T186K

Frequency of potentially antigenic glycosylation sites in natural H9 isolates

Potential glycosylation site position^a	Mutant(s) in this study^b	Frequency^c
127–129	S127N, T127N	6.8%
131–133	K131N	0.1%
148–150	A150T, L150S	1.5%
188–190	T188N	2.6%
189–191	D189N	0.1%

^aPotential glycosylation sites defined as amino-acid motif N-X-S/T, where X is not P; ^bMutants described in Fig. 1, mutants made in this study shown in bold; ^cTotal frequency of potential glycosylation site at this position, based on full length H9 sequences in the NCBI database as of July 2017

Fig. 2 Sequence variation at antigenically significant H9 haemagglutinin escape mutant sites.

Time-resolved Bayesian HA1 phylogeny and amino-acid identity at each of the 20 mAb escape residues found to significantly affect HI titres with polyclonal chicken antisera. Amino-acid identity is shown by colour, grouped by side-chain property, according to the legend. Each virus (n = 330) included in the phylogeny has associated HI data and was included in integrated modelling of genetic and antigenic data

Amino acids substitutions correlating with antigenic change and corresponding antigenic sites

HA position^a	Described, or equivalent antigenic site			Antigenically distinct amino acids
	H9	H1	H3
72	H9-B	Cb	E	E-G-R
74	UC^b	Cb	E	G-R
121	-^c			I-T
131	UC		A	K-A/I/R/S, A-R
135	Site II	Ca2	A	D-G-N, D-E
150	Site I	Sb	B	A/F/N-V/L, F-S-T
180	-	Sb	B	A-E-T, A-V, E-D
183	Site II/H9-A	Sb	B	N-D/E/T
195	-			A-T
198	-	Ca1	D	D-N
216	Site II	Ca2	D	L-Q
217	H9-A		D	I-Q/T
249	-			A/V-I
264	-		C	K-N
276	-			K-R
288	-		C	I-V
306	-			I-K

^aH9 mature numbering used throughout; ^bUC indicates known antigenic residue (by escape mutant residue) in H9 not previously categorised into a specific antigenic site; ^cdash indicates site not previously described as being antigenic

Fig. 3 Heatmap showing the impacts of reciprocal amino-acid substitutions introduced at sites identified by modelling.

Changes in HI titre (log₂) associated with each introduced substitution are shown by heatmap, measured using antiserum both lacking the introduced substitution (parent-like) and possessing the introduced mutation (mutant-like) from combinations of the viruses UDL1/08, Em/R66 and HK/33982. Changes in HI titre associated with each pair of substitutions were interpreted as effects of altered avidity and antigenicity using Equation 1, are also shown. Avidity effects cause an increase (blue) or drop (orange) in titre depending on the direction of substitution while antigenic effects are the estimated change in titre resulting from antigenic dissimilarity. X indicates not individually significant

Fig. 4 Antigenic structure of H9 HA.

Homotrimers of H9 HA. Selected receptor-binding residues shown in red (P92, G128, T129, S130, S131, A132, W142, N173, L184, Y185, N214, G215, G218 and R219). Images made in Pymol⁵² (Schrödinger) based on the structure of A/swine/Hong Kong/9/1998 (Protein databank ID:1JSD)⁵³. a Residues recognised by mouse mAbs, positions with updated site H9-A shown in green, H9-B residues shown in blue. b Residues labelled with substitutions that affect the binding of chicken polyclonal antisera. Non-glycosylation altering substitutions shown in yellow; glycosylation site adding mutations shown in black; site 150, which had both glycosylation adding and non-glycosylation adding mutations shown in brown

Summary of residues investigated in this study and their relation to antigenicity and avidity

The PyMOL Molecular Graphics System, Version 1.3r1 (2010).

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HaYStevensDJSkehelJJWileyDCX-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogsProc. Natl. Acad. Sci. USA200198111811118610.1073/pnas.201401198

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