We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014–2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example, H7N9. In recent years it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses. Importance Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this ‘fitness'. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions – however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to either reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focussing particularly on the natural avian host, chickens. We found H9N2 PA-X induced robust host shutoff in both mammalian and avian cells and increased replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.