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

Abstract

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses.

H9 Influenza Viruses: An Emerging Challenge

“…The Eurasian H9 HA sequences fall into three major sublineages: h9.2, h9.3, and h9.4. The emergence of the poultry-adapted H9 IAVs in China has promoted great phylogenetic diversity giving rise to many clades/subclades (Guo et al 2000;Peacock et al 2019). The most prevalent clades are named after their prototypic viruses: Y439-h9.2 (A/duck/Hong Kong/Y439/ 1997), BJ94-h9.3 (A/chicken/Beijing/1/94), and G1-h9.4 (A/quail/Hong Kong/G1/1997).…”

“…Viruses circulating in the wild bird reservoir in Eurasia vary by geographic location but, generally, fall into the Y439-h9.2 lineage (also referred to as Korean lineage) (Figs. 2 and 3; Dalby and Iqbal 2014;Peacock et al 2019). Y439-like viruses were initially identified from disease outbreaks in chickens in South Korea and from domestic ducks in Hong Kong in the mid-1990s.…”

“…Y439-like viruses were initially identified from disease outbreaks in chickens in South Korea and from domestic ducks in Hong Kong in the mid-1990s. Since then, Y439-h9.2-like viruses have been isolated sporadically from wild birds and have been associated with outbreaks in domestic birds in Europe, Indonesia, Southeast Asia, Southeast Russia, Oceania, South Africa, Zambia, and Malawi (Abolnik et al 2006;Bonfante et al 2018;Peacock et al 2019). In Europe, vaccination has been adopted to prevent reoccurrences especially in turkeys in Germany and Italy (Capua and Alexander 2009).…”

“…These viruses can be further subdivided based on relatedness and geographic distribution as "Eastern" (G1-h9.4.1) and "Western" (G1-h9.4.2) sublineages ( Fig. 1; Dong et al 2011;Fusaro et al 2011;Nagy et al 2017;Peacock et al 2019). The G1-h9.4.1 strains are mainly found in South China, Vietnam, and Cambodia, and are mostly associated to minor poultry species such as quail, partridges, and guinea fowls.…”

“…Asian-origin H9N2 IAVs pose a threat to public health as zoonotic human infections are being reported in China, Hong Kong, Bangladesh, Egypt, Pakistan, and Oman (Table 2; Guan et al 1999;Cameron et al 2000;Butt et al 2005;Banet-Noach et al 2007;Jia et al 2009;Fusaro et al 2011;Blair et al 2013;Monne et al 2013;Yu et al 2013;Shanmuganatham et al 2014;Huang et al 2015;Sun and Liu 2015;Wang et al 2015b). As of June 2019, there have been 59 laboratory-confirmed H9 infections in humans with approximate equal distribution between sexes ( public databases show sequence information for 29 of those cases) (Peacock et al 2019). Young children, <8 years of age, were the most frequently affected.…”