H9N2 Hong-Kong

What is the history of H9N2 virus detection in humans in Hong Kong?

H9N2 avian influenza viruses have caused repeated human infections in Hong Kong since the late 1990s. In 1999, two H9N2 viruses were isolated from humans in Hong Kong for the first time, drawing worldwide attention from health authorities . Later, in November 2003, another H9N2 avian influenza virus was isolated from a 5-year-old child with flu-like illness in Hong Kong . This 2003 case (A/Hong Kong/2108/03) demonstrated that H9N2 viruses continued to pose a threat to human health. Earlier, a separate H9N2 virus (GZ/333/99) had caused disease in a 22-month-old girl who developed fever, cough, and viral bronchitis in 1999 . Serological studies during the late 1990s showed that the seroprevalence of H9 antibodies in southern China was at least 2% among the human population, suggesting more widespread infections than clinically detected cases .

How do researchers classify H9N2 viruses found in Hong Kong?

Researchers classify H9N2 viruses through:

• Genotyping: The human isolate from 2003 (HK/2108/03) belongs to genotype A of H9N2 influenza viruses . Phylogenetic and antigenic analyses of H9N2 viruses isolated from Hong Kong markets suggest three distinct sublineages .

• Source Analysis: The viruses are categorized based on their host origin (chickens, quail, other birds) and geographic location.

• Genetic Lineage Assessment: Molecular characterization of H9N2 viruses has revealed that those isolated from Eurasia and America belong to different genetic lineages. Among the chicken H9N2 viruses in Hong Kong, six gene segments were apparently derived from earlier chicken H9N2 viruses isolated in China, while the PB1 and PB2 genes are closely related to H5N1 viruses and a quail H9N2 virus (A/quail/Hong Kong/G1/97) .

• Mutation Grouping: Researchers have categorized H9N2 viruses based on key mutations in their binding sites, particularly those affecting receptor binding preferences .

What genetic reassortment events have been identified in Hong Kong H9N2 viruses?

Genetic reassortment of H9N2 viruses in Hong Kong involves complex patterns of gene exchange:

• H9N2-H5N1 Reassortment: Evidence from genomic analysis has demonstrated that H9N2 viruses have provided genes for the H5N1 and H7N9 subtypes that have infected mammals and posed threats to human health . Many of the 1997 chicken H9 isolates from Hong Kong markets were reassortants, with six gene segments apparently derived from earlier chicken H9N2 viruses isolated in China, while the PB1 and PB2 genes were closely related to those of the H5N1 viruses and the quail H9N2 virus A/quail/Hong Kong/G1/97 .

• Quail Virus as Gene Donor: The similarity of the internal genes of A/quail/Hong Kong/G1/97 virus to those of the H5N1 influenza viruses suggests that this quail virus may have been the internal gene donor for the human and poultry H5N1 influenza viruses in Hong Kong in 1997 .

• Multiple Genotype Generation: Antigenic and genetic analyses revealed that H9N2 influenza viruses have continued to evolve and reassort with other influenza viruses of avian origin to generate multiple genotypes . This ongoing evolution is documented in the continuing research on H9N2 avian influenza viruses in poultry in southern China .

How have receptor binding properties of H9N2 viruses evolved in Hong Kong isolates?

The evolution of receptor binding properties in H9N2 viruses from Hong Kong demonstrates adaptation toward enhanced human receptor recognition:

• Shift in Receptor Preference: Some H9N2 influenza viruses currently circulating in southern China have acquired molecular features that allow them to preferentially bind to α-2,6-NeuAcGal receptors, which are predominant in human upper respiratory tracts . This receptor specificity shift increases their potential for human infection.

• Key Mutations Affecting Binding: Research using docking simulation tools has categorized H9N2 hemagglutinin mutations according to their effects on ligand-binding interactions . These mutations can be divided into those involved in direct ligand-binding interaction and those involved in indirect interaction, with the 200-loop and 190-Helix acting as intermediate elements that regulate the effect of mutations on LSTc-binding .

• Binding Pocket Adaptability: Calculations indicate that all studied H9N2 viruses can establish tight binding with human receptors (LSTc) although mutations cause various perturbations to the local conformation of the binding pocket. A marginal equilibrium appears to exist between the conservative ligand-receptor interaction and the conformational dynamics of the binding pocket, potentially allowing the virus to accommodate mutations to adapt to various environments .

What evidence exists for avian-to-human transmission of H9N2 in Hong Kong?

Several lines of evidence support avian-to-human transmission of H9N2 viruses in Hong Kong:

• Direct Genetic Relationship: Genetic and antigenic analyses showed that the counterpart of the human H9N2 isolate from 2003 (HK/2108/03) was present in live poultry markets of Hong Kong during the same period . This virus was of purely avian origin and closely related to some viruses circulating in poultry in the markets of Hong Kong.

• No Direct Contact Required: In the case of the 5-year-old child infected in 2003, there was no known direct contact with poultry before the onset of infection . Similarly, neither the 22-month-old baby infected with GZ/333/99 in 1999 nor her mother (who also showed serological evidence of infection) had a history of contact with animals . This suggests that transmission could occur through indirect contact with poultry or potentially from another human.

• Experimental Evidence: Research has shown that some H9N2 isolates can cause respiratory infection in experimental mammalian models like Syrian hamsters, suggesting efficient replication in mammalian hosts .

What methodologies are used to assess the pandemic potential of H9N2 viruses?

Researchers employ multiple approaches to evaluate the pandemic potential of H9N2 viruses:

• Receptor Binding Studies: Docking simulation tools are used to investigate interactions between H9N2 hemagglutinin and mammalian receptors (like α-2,6-linked lactoseries tetrasaccharide c) at the atomic level . This helps identify mutations that enhance binding to human-type receptors.

• Animal Models: Experimental infection of Syrian hamsters has been used to demonstrate the ability of H9N2 viruses to replicate efficiently in mammalian hosts . These models help assess virulence and transmission potential.

• Genetic Marker Analysis: Researchers identify and track specific genetic markers associated with increased mammalian adaptation, such as mutations in the receptor binding site of hemagglutinin or adaptations in the polymerase complex .

• Serological Surveillance: Population-based serological studies help determine the extent of previous human infections. Studies in southern China found that 2-2.6% of serum samples from humans contained antibodies to H9 viruses, suggesting more widespread infection than clinically apparent cases .

How has surveillance for H9N2 evolved in Hong Kong poultry markets?

Surveillance systems for H9N2 in Hong Kong poultry markets have developed significantly:

• Initial Focus on Chickens: In 1997, surveillance primarily focused on chickens and other types of poultry, with limited sampling of quail (only 15) . H9N2 viruses were isolated from over 4% of chickens in the live-bird markets of Hong Kong in 1997, but only one of these isolates belonged to the Qa/HK/G1/97-like genotype that possessed internal genes similar to the pathogenic H5N1 viruses of 1997 .

• Expanded Surveillance: Surveillance resumed in 1999 with the objective of determining the prevalence of viruses with the Qa/HK/G1/97 genotype and their distribution among different types of poultry . This expanded surveillance encompassed a broader range of poultry species.

• Systematic Approach: Modern surveillance studies have revealed that H9N2 influenza viruses have been regularly isolated from different types of poultry in live poultry markets in Hong Kong and mainland China . This systematic surveillance helps track the evolution and spread of different viral genotypes.

• Molecular Characterization: Current surveillance incorporates extensive genetic and antigenic characterization of isolates to track the emergence of novel variants and reassortants .

What vaccine development approaches have been tested against H9N2 viruses?

Vaccine development for H9N2 viruses has explored several strategies:

• Whole Virion Vaccines: Researchers have developed whole virion test vaccines with formalin-inactivated egg-grown virus. In experimental studies, intraperitoneal administration of such a vaccine twice to hamsters conferred complete protection against challenge infection by the MDCK cell-grown homologous virus .

• Targeted Protection Assessment: Protection studies focus on evaluating vaccine efficacy against specific circulating strains, particularly those with enhanced human receptor binding or those that are genetically related to viruses that have caused human infections .

• Understanding Antigenic Diversity: Phylogenetic and antigenic analyses have demonstrated the diversity among H9 hemagglutinin proteins . This diversity informs vaccine design to ensure coverage against circulating variants.

How do researchers model receptor-binding properties of H9N2 viruses?

Sophisticated computational and experimental approaches are employed to model H9N2 receptor binding:

• Structural Homology Modeling: Researchers build three-dimensional models of H9N2 hemagglutinin proteins using software packages like Modeller, taking existing crystal structures (such as swine H9N2 influenza virus hemagglutinin, PDB entry code 1JSI) as reference models .

• Molecular Docking Simulations: Tools like Autodock Vina are used to dock receptor analogs (such as LSTc) to the modeled binding pocket in H9N2 hemagglutinin proteins. These simulations typically involve:

  • Grid centering on the sialic acid moiety

  • Assignment of rotatable bonds to the receptor analog

  • Use of algorithms like the Lamarckian genetic algorithm for conformation search

  • Evaluation of binding poses based on RMSD values and weighted scoring functions

• Mutation Effect Analysis: Researchers examine how specific mutations affect binding pocket conformation and interactions with receptors, categorizing mutations based on whether they directly or indirectly influence binding .

What methodologies are used to trace the evolutionary history of H9N2 viruses?

Tracing the evolutionary history of H9N2 viruses involves:

• Phylogenetic Analysis: Researchers construct phylogenetic trees based on gene sequences to determine evolutionary relationships between viral isolates from different time periods, geographic locations, and host species .

• Genotyping: H9N2 viruses are classified into different genotypes based on the constellation of their gene segments. For example, the human H9N2 isolate from 2003 (HK/2108/03) was identified as belonging to genotype A .

• Mutation Pattern Analysis: Key mutations are tracked over time to identify patterns of adaptation and selection. For instance, researchers have identified mutations (like A/V190E and N183H) that alter the binding properties of H9N2 viruses .

• Reassortment Detection: Complex patterns of gene exchange between different influenza subtypes are detected through comparative genomic analysis. This has revealed, for example, that H9N2 viruses have provided genes for H5N1 and H7N9 subtypes with the potential to infect humans .