Use of Highly Pathogenic Avian Influenza A(H5N1) Gain-Of-Function Studies for Molecular-Based Surveillance and Pandemic Preparedness

摘要

INTRODUCTION Zoonotic influenza viruses circulating in poultry and swine pose an ever present threat to human health. In particular, the rapid geographical expansion of highly pathogenic avian influenza (HPAI) A(H5N1) throughout Asia and then into Europe, the Middle East, and Africa during the 2000s galvanized the global community in an attempt to control this rapidly growing threat. Despite successful control efforts in some countries, the virus remains endemic in poultry in at least six countries and continues to cause human illness and deaths as well as countless outbreaks in birds. During the past decade, 668 cases and 393 deaths were detected and reported to the World Health Organization (WHO) ( 1 ). During the 17?years since human infections with HPAI A(H5N1) were first identified in Hong Kong, Special Administrative Region, People’s Republic of China, in 1997, these viruses have evolved substantially through mutation and reassortment, resulting in multiple divergent genotypes and clades ( 2 ). Ongoing H5N1 circulation has appropriately resulted in a focus on sequencing viral genomes to understand the evolution of these viruses and the significance of observed genetic changes. Expanded laboratory capacity for high-throughput Sanger sequencing and recent technological advances, such as next-generation sequencing and parallel computing, have revolutionized the quantity, quality, and availability of gene sequences and our ability to quickly and accurately analyze these data ( 3 ). Consequently, the number of animal and human influenza virus sequences available in publically accessible databases has dramatically increased over the years, as have the bioinformatics tools required for efficient investigation ( 4 , 5 ). These advances in laboratory and analytical methods provide strong incentives to utilize molecular data for pandemic risk assessment of zoonotic influenza viruses at the animal-human interface ( 6 ). However, examination of influenza sequence data alone does not allow us to assess the pandemic potential of a virus. Pandemic risk assessment that utilizes sequence data can take place only after critical genetic signatures are identified through laboratory research into the consequences for relevant biological properties (or phenotypes). These critical genetic features include those that based on previous experimental validation are predicted to confer virulence and/or have the ability to transmit efficiently in mammals. In this context, genomes are sequenced, mutations are detected relative to earlier viruses and prototype strains, significance is appraised based on prior knowledge of genetic markers, and phenotypes are tested using a variety of in vitro and in vivo experiments. Viruses possessing phenotypes of interest or concern often become candidates for reverse-genetics studies, which are essential to elucidate the precise molecular correlate(s) of a given phenotype ( Fig.?1 ). From a molecular epidemiological perspective, this process is at the heart of how the public health community makes informed decisions about the threat posed by zoonotic influenza viruses and which interventions might be most effective ( 7 ). FIG?1? H5N1 or other influenza A virus molecular-based surveillance, gain-of-function research, and pandemic preparedness decision making. Laboratories worldwide have employed reverse genetics to study the mechanisms by which HPAI H5N1 and other zoonotic influenza viruses evolve and how these mechanisms influence host receptor specificity, antigenic variation, replication, pathogenesis, drug susceptibility, and transmission ( 8 – 11 ). Besides being used to create vaccine viruses for the development of live, attenuated ( 12 ) and inactivated prepandemic H5N1 influenza vaccines ( 13 ), reverse-genetics methodologies also have been used for many years to study the phenotypic consequences of particular mutations, including genetic changes that confer a gain of function (GOF). Influenza virus GOF studies have focused on several research areas: in vitro and/or in vivo replication in mammalian cell culture or animal hosts, adaptive mutations conferring changes in host susceptibility, alteration of receptor binding profiles and/or tropism for mammalian airway tissues, enhanced polymerase activity, changes in host antiviral response (e.g., cell signaling pathways), susceptibility to antiviral drugs, and pathogenesis and/or transmissibility in mammalian animal models. Such GOF experiments have elucidated key biological principles and provided the scientific basis for genomic sequence-based risk assessment of zoonotic viruses with pandemic potential. For example, the molecular basis for avian versus mammalian influenza virus receptor binding (α2,3 versus α2,6 sialylated glycans) has been elucidated largely through GOF experiments, and some recent studies that identified specific HA mutations conferring a switch from avian to mammalian host receptor specificity also demonstrated the impa