Assessing the fitness of distinct clades of influenza A (H9N2) viruses

Abstract

Influenza A (H9N2) viruses are a genetically diverse population that infects wild and domestic avian species and mammals and contributed the internal gene segments to the A/H5N1 and A/H7N9 viruses associated with lethal human infections. Here we comprehensively assess the potential risk to mammals of a diverse panel of A/H9N2 viruses, representing the major H9N2 clades, using a combination of in vitro assays (e.g., antiviral susceptibility and virus growth in primary differentiated human airway cells) and in vivo assays (e.g., replication, transmission and/or pathogenicity of viruses in ducks, pigs, mice and ferrets). We observed that viruses isolated from humans, A/Hong Kong/1073/1999 and A/Hong Kong/33982/2009, had the highest risk potential. However, the A/swine/Hong Kong/9A-1/1998 and A/chicken/Hong Kong/G9/1997 viruses also displayed several features suggesting a fitness profile adapted to human infection and transmission. The North American avian H9N2 clade virus had the lowest risk profile, and the other viruses tested displayed various levels of fitness across individual assays. In many cases, the known genotypic polymorphisms alone were not sufficient to accurately predict the virus’ phenotype. Therefore, we conclude that comprehensive risk analyses based on surveillance of circulating influenza virus strains are necessary to assess the potential for human infection by emerging influenza A viruses.

Keywords:

• influenza
• zoonotic disease
• risk assessment
• antivirals
• viral pathogenicity

Acknowledgments

We would like to thank Pamela McKenzie (St Jude Children’s Research Hospital) for administrative support and helpful conversations and Angela McArthur (St Jude Children’s Research Hospital) for manuscript editing. This work was supported by NIH/NIAID Contract HHSN266200700005C (St Jude Center of Excellence for Influenza Research and Surveillance) and American Lebanese Syrian Associated Charities at St Jude.

Supplementary information for this article can be found on Emerging Microbes & Infections' website (http://www.nature.com/EMI/).

Notes

Abbreviations: R, amantadine-resistant virus; S, amantadine-susceptible virus.

NA inhibition assay used 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminicacid (MUNANA) as a substrate at a final concentration of 100 µM. Results are the mean values (IC50, nM) of three experiments±standard deviation (SD).

Susceptibility to adamantanes was based on the presence of a substitution of one of five amino acids (positions 26, 27, 30, 31 and 34) within the transmembrane domain of the M2 protein. Amantadine resistance-associated mutations are shown in parenthesis.

Highest titers, the fastest kinetics indicated by score=4.

Highest titers, the longest duration indicated by score=4.

Seroconversion (SC) in ferrets was determined by HI assay.

1=direct contact (DC) transmission in one animal; 2=DC transmission in two animals; 3=aerosol-contact (AC) transmission in one animal; 4=AC transmission in two animals; 0, no transmission; ND, not done.

Seroconversion (SC) in mice was determined by enzyme-linked immuno sorbent assay.

1=2 ducks shed for 1 day; 2=2 ducks shed for 2 days.

Categories of risk were defined as follows: low=5–8; intermediate (Int) =8–10 or >10 with Q226; high ≥10; very high >10 with L226.