Double mutations in the H9N2 avian influenza virus PB2 gene act cooperatively to increase viral host adaptation and replication for human infections.

Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.

PA Mutations Inherited during Viral Evolution Act Cooperatively To Increase Replication of Contemporary H5N1 Influenza Virus with an Expanded Host Range.

Adaptive mutations and/or reassortments in avian influenza virus polymerase subunits PA, PB1, and PB2 are one of the major factors enabling the virus to overcome the species barrier to infect humans. The majority of human adaptation polymerase mutations have been identified in PB2; fewer adaptation mutations have been characterized in PA and PB1. Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and generally carry the human adaptation PB2-E627K substitution during their dissemination in nature. In this study, we identified other human adaptation polymerase mutations by analyzing phylogeny-associated PA mutations that H5N1 clade 2.2.1 viruses have accumulated during their evolution in the field. This analysis identified several PA mutations that produced increased replication by contemporary clade 2.2.1.2 viruses in vitro in human cells and in vivo in mice compared to ancestral clade 2.2.1 viruses. The PA mutations acted cooperatively to increase viral polymerase activity and replication in both avian and human cells, with the effect being more prominent in human cells at 33°C than at 37°C. These results indicated that PA mutations have a role in establishing contemporary clade 2.2.1.2 virus infections in poultry and in adaptation to infect mammals. Our study provided data on the mechanism for PA mutations to accumulate during avian influenza virus evolution and extend the viral host range.IMPORTANCE Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication in vitro in human cells and in vivo in mice. These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans.

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity.

Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.

PB2 mutations arising during H9N2 influenza evolution in the Middle East confer enhanced replication and growth in mammals.

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks.

Identification of polymerase gene mutations that affect viral replication in H5N1 influenza viruses isolated from pigeons.

Highly pathogenic avian influenza virus H5N1 infects a wide range of host species, with a few cases of sporadic pigeon infections reported in the Middle East and Asia. However, the role of pigeons in the ecology and evolution of H5N1 viruses remains unclear. We previously reported two H5N1 virus strains, isolated from naturally infected pigeons in Egypt, that have several unique mutations in their viral polymerase genes. Here, we investigated the effect of these mutations on H5N1 polymerase activity and viral growth and identified three mutations that affected viral polymerase activity. The results showed that the PB1-V3D mutation significantly decreased polymerase activity and viral growth in both mammalian and avian cells. In contrast, the PB2-K627E and PA-K158R mutations had moderate effects: PB2-K627E decreased and PA-K158R increased polymerase activity. Structural homology modelling indicated that the PB1-V3D residue was located in the PB1 core region that interacts with PA, predicting that the PB1 mutation would produce a stronger interaction between PB1 and PA that results in decreased replication of pigeon-derived H5N1 viruses. Our results identified several unique mutations responsible for changes in polymerase activity in H5N1 virus strains isolated from infected pigeons, emphasizing the importance of avian influenza surveillance in pigeons and in studying the possible role of pigeon-derived H5N1 viruses in avian influenza virus evolution.

Genetic characterization of highly pathogenic avian influenza H5N1 viruses isolated from naturally infected pigeons in Egypt.

Avian influenza viruses impose serious public health burdens with significant mortality and morbidity not only in poultry but also in humans. While poultry susceptibility to avian influenza virus infection is well characterized, pigeons have been thought to have low susceptibility to these viruses. However, recent studies reported natural pigeon infections with highly pathogenic avian influenza H5N1 viruses. In Egypt, which is one of the H5N1 endemic areas for birds, pigeons are raised in towers built on farms in backyards and on house roofs, providing a potential risk for virus transmission from pigeons to humans. In this study, we performed genetic analysis of two H5N1 virus strains that were isolated from naturally infected pigeons in Egypt. Genetic and phylogenetic analyses showed that these viruses originated from Egyptian H5N1 viruses that were circulating in chickens or ducks. Several unique mutations, not reported before in any Egyptian isolates, were detected in the internal genes (i.e., polymerase residues PB1-V3D, PB1-K363R, PA-A369V, and PA-V602I; nucleoprotein residue NP-R38K; and nonstructural protein residues NS1-D120N and NS2-F55C). Our findings suggested that pigeons are naturally infected with H5N1 virus and can be a potential reservoir for transmission to humans, and showed the importance of genetic analysis of H5N1 internal genes.