The enzootic and zoonotic nature of H9N2 avian influenza viruses poses a persistent threat to the global poultry industry and public health. In particular, the emerging sublineage h9.4.2.5 of H9N2 viruses has drawn great attention. In this study, we determined the effects of the flexibility at residues 226 and 227 in the hemagglutinin on the receptor avidity and immune evasion of H9N2 viruses. The solid-phase direct binding assay showed that residue 226 plays a core role in the receptor preference of H9N2 viruses, while residue 227 affects the preference of the virus for a receptor. Consequently, each of these two successive residues can modulate the receptor avidity of H9N2 viruses and influence their potential of zoonotic infection. The antigenic map based on the cross-hemagglutination inhibition (HI) titers revealed that amino acid substitutions at positions 226 or 227 appear to be involved in antigenic drift, potentially resulting in the emergence of H9N2 immune evasion mutants. Further analysis suggested that increased receptor avidity facilitated by residue 226Q or 227M resulted in a reduction in the HI titer. Among the four naturally-occurring amino acid combinations comprising QQ, MQ, LQ, and LM, the number of viruses with LM accounted for 79.64% of the sublineage h9.4.2.5 and the rescued virus with LM exhibited absolute advantages of in vitro and in vivo replication and transmission. Collectively, these data demonstrate that residues 226 and 227 are under selective pressure and their synergistic regulation of receptor avidity and antigenicity is related to the evolution of circulating H9N2 viruses.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40μg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

Infectious bronchitis (IB) is a major threat to the global poultry industry. Despite the availability of commercial vaccines, the IB epidemic has not been effectively controlled. The exploration of novel IBV vaccines may provide a new way to prevent and control IB. In this study, BLP-S1, a bacterium-like particle displaying the S1 subunit of infectious bronchitis virus (IBV), was constructed using the GEM-PA surface display system. The immunoprotective efficacy results showed that BLP-S1 can effectively induce specific IgG and sIgA immune responses, providing a protection rate of 90% against IBV infection in 14-day-old commercial chickens. These results suggest that BLP-S1 has potential for the development of novel vaccines with good immunogenicity and immunoprotection.

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Nipah virus (NiV) causes severe, lethal encephalitis in humans and pigs. However, there is no licensed vaccine available to prevent NiV infection. In this study, we used the reverse genetic system based on the attenuated rabies virus strain SRV9 to construct two recombinant viruses, rSRV9-NiV-F and rSRV9-NiV-G, which displayed the NiV envelope glycoproteins F and G, respectively. Following three immunizations in BALB/c mice, the inactivated rSRV9-NiV-F and rSRV9-NiV-G alone or in combination, mixed with the adjuvants ISA 201 VG and poly (I:C), were able to induce the antigen-specific cellular and Th1-biased humoral immune responses. The specific antibodies against rSRV9-NiV-F and rSRV9-NiV-G had reactivity with two constructed bacterial-like particles displaying the F and G antigens of NiV. These data demonstrate that rSRV9-NiV-F or rSRV9-NiV-G has the potential to be developed into a promising vaccine candidate against NiV infection.