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

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.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity

“…Conversely, the unglycosylated epitopes of rSL20(P)-MVV310PB2 may be the major target of humoral immunity, but the antibody cannot bind to the epitopes of r310-NS28 due to the steric hindrance of the N-glycans. The result supports the importance of the epitopes and the preferred acquisition of 144N or 158N glycans to evade vaccine-induced antibodies by clade 2.3.2.1c H5N1 viruses [63]. A limitation of our study was that the inactivated virus antigens were not assayed by HAU before the vaccine was administered as a vaccine.…”

“…SL20wt is classified into the genotype S H9N2 viruses in China along with N20-99 and SD1844 [33,37]. It shares the common MPMs found in most genotype S H9N2 viruses, including I292V and V598I in the PB2 gene, Q226L in the HA gene, a 3 amino acid deletion (63)(64)(65) in the stalk region of NA, N30D, and T215A in the M1 gene, and P42S and a C-terminal truncation from 237 to 217 amino acids in the NS1 gene, etc. (Table S2) [15,[24][25][26][38][39][40][41][42][43][44][45][46].…”

Non-Mouse-Adapted H1N1pdm09 Virus as a Model for Influenza Research

“…Highly pathogenic avian influenza viruses displayed a higher thermostability compared with viruses with low pathogenicity [ 68 ]. The resistance of HA to high temperatures contributes to environmental resistance in viruses [ 69 ] and affects pathogenicity in sensitive mammalian hosts [ 70 ]. Pathogenicity for animals of the SA may be associated with more pronounced thermostability of its HA.…”

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift

“…Interestingly, phenotypic attributes conferred by one amino acid substitution may not be conferred if another amino acid was selected for at the subsite. Although an American-lineage R144G (H5: R140G; H7: R133G) escape mutant described above had increased affinity to numerous α-2,3 receptor analogues, subsequent phenotypic characterization of a gs/Gd-lineage escape mutant containing the N144S substitution (H5: N140S; H7: N133S) had reduced binding to 3′SLN-PAA receptor analogue and to chicken erythrocytes, and exhibited a reduction in thermostability compared the parental strain (An et al 2019 ). Another study characterizing Eurasian non-gs/Gd-lineage escape mutants containing S145P/Y substitutions (H5: 141; H7: 134) demonstrated that HA thermostability of escape mutants was dependent on this amino acid (Timofeeva et al 2020a ), and HI activity has been shown to be modulated by the amino acid present at subsite 57 (immature protein: 65) (Henry Dunand et al 2016 ).…”