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Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential. Author summary: Avian influenza viruses (AIV) including viruses of the H5Nx Goose/Guangdong-lineage caused hundreds of human infections and fatalities worldwide and pose a continuous zoonotic and pandemic threat. It is important to understand the role of mutations that support AIV-infection in mammals after bird-to-human transmission. Here, we describe the prevalence and the role of mutations in the viral neuraminidase (NA), which is mainly responsible for virus release from infected cells, preferentially selected in human H5N1 viruses. Compared to their avian ancestors, human-like H5N1 had less NA in virus particles and exhibited lower NA activity conferred mainly by the L204M substitution. Furthermore, NA mutations increased the binding of H5N1 to human-type receptors, acted synergistically to confer high virus replication in avian and human cells and/or modulated virulence and replication in mice, but not in chickens. This study is important to understand the genetic and biological properties of human AIV and will help to predict zoonotic potential AIV. [ABSTRACT FROM AUTHOR]

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry. [ABSTRACT FROM AUTHOR]

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa, and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk, we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA, and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA, and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals. [ABSTRACT FROM AUTHOR]

The main findings of the post-mortem examination of poultry infected with highly pathogenic avian influenza viruses (HPAIV) include necrotizing inflammation and viral antigen in multiple organs. The lesion profile displays marked variability, depending on viral subtype, strain, and host species. Therefore, in this study, a semiquantitative scoring system was developed to compare histopathological findings across a wide range of study conditions. Briefly, the severity of necrotizing lesions in brain, heart, lung, liver, kidney, pancreas, and/or lymphocytic depletion in the spleen is scored on an ordinal four-step scale (0 = unchanged, 1 = mild, 2 = moderate, 3 = severe), and the distribution of the viral antigen in parenchymal and endothelial cells is evaluated on a four-step scale (0 = none, 1 = focal, 2 = multifocal, 3 = diffuse). These scores are used for a meta-analysis of experimental infections with H7N7 and H5N8 (clade 2.3.4.4b) HPAIV in chickens, turkeys, and ducks. The meta-analysis highlights the rather unique endotheliotropism of these HPAIV in chickens and a more severe necrotizing encephalitis in H7N7-HPAIV-infected turkeys. In conclusion, the proposed scoring system can be used to condensate HPAIV-typical pathohistological findings into semiquantitative data, thus enabling systematic phenotyping of virus strains and their tissue tropism. [ABSTRACT FROM AUTHOR]

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry. [ABSTRACT FROM AUTHOR]