Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets - PubMed (original) (raw)

. 2011 Aug 23;108(34):14264-9.

doi: 10.1073/pnas.1111000108. Epub 2011 Aug 8.

Chi-Hui Liang, Chung-Yi Wu, Heather L Forrest, Angela Ferguson, Ka-Tim Choy, Jeremy Jones, Diana Dik-Yan Wong, Peter Pak-Hang Cheung, Che-Hsiung Hsu, Olive T Li, Kit M Yuen, Renee W Y Chan, Leo L M Poon, Michael C W Chan, John M Nicholls, Scott Krauss, Chi-Huey Wong, Yi Guan, Robert G Webster, Richard J Webby, Malik Peiris

Affiliations

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

Hui-Ling Yen et al. Proc Natl Acad Sci U S A. 2011.

Abstract

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Transmissibility of seasonal H3N2 (A), pandemic H1N1 (B), precursor swine (C), and reassortant swine (D) influenza viruses in ferrets. Values are virus titers (log10TCID50/mL) in the nasal washes of individual donor ferrets (yellow), direct contacts (green), and respiratory-droplet contacts (blue). D shows experiments for sw915 in two separate panels. The detection limit was 101.5 TCID50/mL.

Fig. 2.

Fig. 2.

Polymerase activity and replication efficiency of the recombinant viruses in vitro. (A) The transcription and replication efficiency of the polymerase complex of sw915 and HK415742 were evaluated at 37 °C or 33 °C. Values are the mean ± SD from two replicates from independently repeated experiments. (B) Viral replication kinetics in MDCK or differentiated NHBE cells were determined at 37 °C at multiplicity of infection (MOI) = 0.001 or 0.01, respectively. Log10 mean ± SD values from three replicates (MDCK) or two or three independently performed experiments (NHBE) are shown.

Fig. 3.

Fig. 3.

Transmissibility of the recombinant sw915 viruses in ferrets. The direct-contact and respiratory-droplet transmissibility of RG-sw915 (A), RG-sw915xHK415742NA (B), and RG-sw915xHK415742PB2,PA,HA,NA (C) viruses in ferrets. Viral titers (log10TCID50/mL) were obtained from the nasal washes of individual donor ferrets (yellow), direct contacts (green), and respiratory-droplet contacts (blue). The detection limit was 101.5 TCID50/mL.

Fig. 4.

Fig. 4.

HA glycan array and NA enzyme kinetics. (A) Glycan array analysis of receptor-binding specificity against sialyl glycans (

Fig. S1

). (B) Binding intensity of glycans 23, 27, 28, and 29 with 3 × 106 pfu/mL of HK415742 and sw915 viruses. (C) NA enzyme kinetics of recombinant PR8, PR8xHK415742NA, PR8xsw915NA, PR8xswNS29NA, and PR8xswAR2976NA viruses using 3′SL or 6′SL as substrates. The kinetic data were fit to the Michaelis–Menten equation by nonlinear regression to determine the Michaelis constant (_K_M) and maximum velocity (_V_max) of substrate conversion. Transmission electron microscopy shows virus aggregates in MDCK cells infected with RG-sw915 virus (D) but not in MDCK cells infected with RG-sw915xHK415742NA virus (E) (MOI = 0.1 and fixed after 12 h).

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