Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

Abstract

Abstract: Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

Keywords: HA trimer stability; clade 2.3.2.1c H5N1 virus; immunity evasion; mammalian pathogenicity; thermostability.

Figure 1

Growth kinetics of recombinant H5N1 viruses in MDCK and A549 cells. Each recombinant virus was diluted to 10⁵ 50% chicken embryo infection dose (EID₅₀)/0.1 mL, and 0.5 mL of diluents were inoculated into confluent (a) MDCK and (b) A549 cells in 6-well plates for 1 h. After 1 h, the inoculated virus was removed, and 1 mL of fresh medium was added. During 72 h of incubation, the supernatant was harvested at 0, 24, 48, and 72 hpi, and 50% tissue culture infectious dose (TCID₅₀)/0.1 mL of each time point was measured in MDCK cells. The TCID₅₀/0.1 mL values are the average of three independent experiments. #, *, significant differences of rH5N1-N144S-I223V (#) and rPR8 (*) in comparison with other viruses (p < 0.05).

Figure 2

Mouse pathogenicity of recombinant H5N1 viruses. (a) Mortality and (b) weight loss of mouse experimental groups infected with recombinant H5N1 viruses. Five six-week-old female BALB/c mice per group were inoculated with 10⁶ EID₅₀ of virus or an equivalent volume PBS (mock) intranasally. Weight loss was monitored for 2 weeks, and mice with more than 20% weight loss were euthanized. The weight loss was calculated based on the body weight measured at 0 dpi, and the data are the average of each group; * significant difference of the rH5N1-I223V and rH5N1-N144S groups compared to the mock groups (p < 0.05).

Figure 3

Receptor binding affinity of recombinant H5N1 viruses. The two types of serially diluted biotinylated sialylglycopolymers (Neu5Acα2-3Galb1-4GlcNAcb–PAA-biotin (3′SLN–PAA) and Neu5Acα2-6GalNAca–PAA-biotin (6′SLN–PAA)) were incubated with the same concentration (10⁵ EID₅₀) of recombinant viruses. After development with horseradish peroxidase (HRP)-conjugated streptavidin and 3,3’5,5’-Tetramethylbenzidine (TMB) substrate, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured. (a) Receptor binding affinity of recombinant viruses to 3′SLN–PAA and (b) Receptor binding affinity of recombinant viruses to 6′SLN–PAA. The absorbance data are the average of three independent experiments, # significant difference of rH5N1 and rH5N1-N144S-I223V compared to the other viruses, * significant difference compare to rH5N1–N144S (p < 0.05).

Figure 4

Heat stability of recombinant H5N1 viruses. Each of the recombinant viruses was diluted to a 2⁴ HA titer, and aliquots were incubated at 60 °C for 0, 5, 15, and 30 min. After heat treatment, the HA titer of each aliquot was measured by HA assay with 1% chicken RBCs.

Figure 5

Location and intermolecular interaction of 144N and 223V/I residues in the 3D structure of the HA trimer. HA and HA trimer structure were modified from 4juk.pdb and 6e7g.pdb using PyMOL. (a) 223I and (c) 223V were located in the 220-loop of the receptor binding site (RBS) of the globular head. Position 223 was close to position 226Q, and both 223I and 223V interacted with 226Q by hydrogen bond (dotted line). (a), (c) 144N followed by 145S and 146S were located near the RBS, and 144N glycosylation was formed by N-X-S/T. (b) 223I and (d) 223V were close to the 207S of another HA monomer, and 223I had more side chains extruding and was much closer to 207S than 223V.

Figure 6

Verification of 144N-glycosylation by Western blotting. Recombinant viruses untreated and treated with PNGase F enzyme were denatured and separated by SDS-PAGE. Transferred membranes were incubated with rabbit anti-influenza A H5N1 (A/Vietnam/1194/2004) HA IgG, followed by goat anti-rabbit IgG HRP-conjugated secondary antibody. Then, HRP was developed by TMB substrate. HA proteins of rH5N1 and rH5N1-I223V had 144N-glycan, and they had higher molecular weight than rH5N1-N144S and rH5N1-N144S-I223V in the absence of PNGase F enzyme treatment (−). However, the difference disappeared after treatment of PNGase F enzyme (+).