220 mutation in the hemagglutinin of avian influenza A (H7N9) virus alters antigenicity during vaccine strain development

Figures & data

Figure 1. Modified sequence in HA and NA of reassortant viruses. (A) HA cleavage site sequence. LAPI–HA7: consensus sequence of low pathogenic avain influenza viruses H7 (reference to the sequence in NCBI gene bank, Accession Number: APR73185, AJU15335, AKI82233, APW83929, AHL21384, AJE61993, ANH96462, ALR82230, AJU15321, APW83918); RG-17SF003HA: sequence of (6+2) reassortant H7N9 virus by reverse technology with deletion of KRTA; Wild-17SF003HA: sequence of wild-type virus A/Guangdong/17SF003/2016; ↓: proteolytic cleavage site. (B) Part of amino acid sequence associated with resistance to neuraminidase inhibitors (N2 numbering: 291–293 position). Wild—17SF003NA: wild-type 17SF003 virus containing NA-292K/R; RG-17SF003NA: (6+2) reassortant 17SF003 virus containing NA-292R. Change sequences were in bold.

Table 1. Amino acid difference and virus titers of reassortant viruses.

Viruses	Passage history^a	Amino acid residue at 220 position of HA (%)^b	HA titer	logEID50/ml	LogTCID50/ml
RG-SF003	V1E1	R/G/I(100,0,0)	512	9.0	7.3
	V1E2	R/G/I(99,1,0)	512	—^c	—
	V1E3	R/G/I(88,10,2)	512	—	—
	V1E4	R/G/I(35/50/15)	1024	—	—
	V1E5	R/G/I(22/70/8)	1024	9.22	7.87
PR8	C1E2	R	1024	9.44	8.0

^a ‘V’ Vero cells; ‘E’ eggs;’ C’ MDCK cells.

^b H3 HA numbering system was used.%: The percentage of different amino acids.

^c ‘-’ denote not done. All results were determined by haemagglutination assay and calculated by the Reed–Muench method.1% turkey erythrocytes were used.

Table 2. Antigenic analysis of candidate vaccine viruses by hemagglutination inhibition.

	Ferret antisera
Antigen	AH1	AH1-RG	SH2	SH2-RG	HN-RG	SF003-RGV1E1	SF003-RGV1E5
A/Anhui/1/2013	80	160	640	80	320	160	320
A/Anhui/1/2013-RG	160	320	640	320	320	320	640
A/Shanghai/2/2013	160	160	640	160	320	160	640
A/Shanghai/2/2013-RG	160	160	1280	320	320	320	640
A/Hunan/02650/2016	80	320	320	80	320	640	640
A/Hunan/02650/2016-RG	80	320	320	40	320	320	640
A/Guangong/17SF003/2016	<20	20	80	<20	<20	80	80
A/Guangong/17SF003/2016-RG V1E1	<20	20	80	<20	<20	80	80
A/Guangong/17SF003/2016-RG V1E5	80	320	320	80	320	640	1280

AH1:A/Anhui/1/2013(wildtype); AH1-RG:A/Anhui/1/2013 (reverse genetics);SH2:A/Shanghai/2/2013 (wildtype); SH2-RG: A/Shanghai/2/2013(reverse genetics);HN-RG:A/Hunan/2650/2016(reverse genetics), SF003-RGV1E1: A/Guangong/SF003/2016(reverse genetics, initial rescued in Vero cells followed by one passages growth in eggs); SF003-RGV1E5: A/Guangong/SF003/2016(reverse genetics, initial rescued in Vero cells followed by five passages growth in eggs).

Bold font indicates homologous.

Figure 2. Growth characteristics of different viruses in MDCK cells. V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs; PR8: A/Puerto Rico/8/34 generated by RG. MDCK cell monolayers were infected at a multiplicity of infection of 0.001 of different viruses. The data was the results of three independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0). ** represents p < 0.01; * represents p < 0.05.

Figure 3. Replication of reassortant viruses in MDCK cells with or without TPCK-trypsin. V1E1: RG-17SF003V1E1 with initial growth in Vero cells followed by 1 passage in eggs; V1E5: RG-17SF003V1E5 with initial growth in Vero cells followed by 5 passages in eggs; PR8: A/Puerto Rico/8/34 generated by RG. The original viruses were diluted from 10⁻¹to 10⁻⁵ and each diluted viruses were inoculated in MDCK cells. Mock cells were inoculated with PBS.

Table 3. Virulence assessment of RG-SF003 in chicken embryos and chickens.

viruses	CELD50 ^a (logEID50/ml)	Mortality^b (death/total)	IVPI^c
RG-SF003V1E1	≥9.0	0/10	0
RG-SF003V1E5	≥9.22	0/10	0

^a CELD₅₀:50% chicken embryo lethal dose.

^b Chickens were examined at 24-hour intervals for 10 days.

^c IVPI denote intravenous pathogenicity index.

Figure 4. A Total protein content (left Y axis) and HA proportion (right Y axis). Total protein content per 10 eggs of each virus. HA proportion was determined using the following equation: HA1+HA2 /HA1+HA2+NP+M. B SDS-PAGE profile of various influenza viruses using the same sample amount. M: Marker (KD). PR8: A/Puerto Rico/8/34 generated by RG; V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs. The data was the results of two independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0).** represents p < 0.01; *** represents p < 0.001.