Amino acid substitutions in the H5N1 avian influenza haemagglutinin alter pH of fusion and receptor binding to promote a highly pathogenic phenotype in chickens

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.

Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity

Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996–1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence.

VAKSIN FLU BURUNG DI MANUSIA SEBAGAI UPAYA ALTERNATIF PENCEGAHAN TERHADAP RE-EMERGING VIRUS H5N1

A total of 182 human cases of avian influenza have been reported in Indonesia in the period 2004-2011, with a case fatality rate (CFR) among them of 86%. Various risk factors for avian influenza transmission have also been identified, including a high population, having many types of poultry (broiler, layer, village chickens, etc), traditional poultry systems that promote close human relationships, and the presence of several areas in Indonesia which are strategic locations as a place to rest and meet various wild birds. Vaccination is one of the efforts to prevent the re-emergence of H5N1 avian influenza transmission. However, the avian influenza vaccine for the public is not yet available in Indonesia. The purpose of this review article is to examine the avian influenza vaccine in humans as a preventive measure against the re-emergence of avian influenza in Indonesia, and to determine the progress of developing avian influenza vaccines. The results of the review show that several avian influenza vaccine candidates have been approved by WHO, one of which is a vaccine candidate using an isolated virus from Indonesia. This information on the development of avian influenza vaccines for humans is useful and can be used as an avian influenza pre-pandemic preparedness in Indonesia. Abstrak Sebanyak 182 kasus flu burung pada manusia telah dilaporkan di Indonesia pada periode tahun 2004-2011, dengan case fatality rate (CFR) sebesar 86%. Berbagai faktor risiko penularan flu burung juga telah diidentifikasi, antara lain jumlah penduduk yang tinggi, memiliki banyak jenis unggas (ayam broiler, layer, kampong, dsb), sistem perunggasan tradisional yang membuat kedekatan hubungan antara manusia, dan adanya beberpa wilayah di Indonesia yang menjadi lokasi strategis sebagai tempat istirahat dan bertemunya berbagai unggas liar. Vaksinasi merupakan salah satu upaya untuk mencegah munculnya kembali penularan flu burung H5N1. Namun demikian, vaksin flu burung untuk masyarakat belum tersedia hingga saat ini. Tujuan dari review artikel ini adalah untuk mengkaji vaksin flu burung pada manusia sebagai tindakan pencegahan terhadap kemunculan kembali flu burung di Indonesia, dan untuk mengetahui kemajuan pengembangan vaksin flu burung. Hasil review menunjukkan beberapa kandidat vaksin flu burung telah disetujui oleh WHO, salah satunya adalah kandidat vaksin menggunakan isolat virus asal Indonesia. Informasi perkembangan vaksin flu burung bagi manusia ini bermanfaat dan dapat digunakan sebagai kesiapsiagaan prepandemi flu burung di Indonesia.

Viral–Host Interactome Analysis Reveals Chicken STAU2 Interacts With Non-structural Protein 1 and Promotes the Replication of H5N1 Avian Influenza Virus

As a highly pathogenic influenza virus, H5N1 avian influenza virus (AIV) poses a great threat to poultry production and public health. H5N1 AIV has a small genome and, therefore, relies heavily on its host cellular machinery to replicate. To develop a comprehensive understanding of how H5N1 AIV rewires host cellular machinery during the course of infection, it is crucial to identify which host proteins and complexes come into physical contact with the viral proteins. Here, we utilized affinity purification mass spectrometry (AP-MS) to systematically determine the physical interactions of 11 H5N1 AIV proteins with host proteins in chicken DF1 cells. We identified with high confidence 1,043 H5N1 AIV–chicken interactions involving 621 individual chicken proteins and uncovered a number of host proteins and complexes that were targeted by the viral proteins. Specifically, we revealed that chicken Staufen double-stranded RNA-binding protein 2 interacts with AIV non-structural protein 1 (NS1) and promotes the replication of the virus by enhancing the nuclear export of NS1 mRNA. This dataset facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of H5N1 AIV infection.