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. 2009 May 5;106(18):7565-70.
doi: 10.1073/pnas.0900877106. Epub 2009 Apr 20.

Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus

Erin M Sorrell  1 , Hongquan Wan, Yonas Araya, Haichen Song, Daniel R Perez
Affiliations

Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus

Erin M Sorrell et al. Proc Natl Acad Sci U S A. .

Abstract

Pandemic influenza requires interspecies transmission of an influenza virus with a novel hemagglutinin (HA) subtytpe that can adapt to its new host through either reassortment or point mutations and transmit by aerosolized respiratory droplets. Two previous pandemics of 1957 and 1968 resulted from the reassortment of low pathogenic avian viruses and human subtypes of that period; however, conditions leading to a pandemic virus are still poorly understood. Given the endemic situation of avian H9N2 influenza with human-like receptor specificity in Eurasia and its occasional transmission to humans and pigs, we wanted to determine whether an avian-human H9N2 reassortant could gain respiratory transmission in a mammalian animal model, the ferret. Here we show that following adaptation in the ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a human H3N2 backbone can transmit efficiently via respiratory droplets, creating a clinical infection similar to human influenza infections. Minimal changes at the protein level were found in this virus capable of respiratory droplet transmission. A reassortant virus expressing only the HA and neuraminidase (NA) of the ferret-adapted virus was able to account for the transmissibility, suggesting that currently circulating avian H9N2 viruses require little adaptation in mammals following acquisition of all human virus internal genes through reassortment. Hemagglutinin inhibition (HI) analysis showed changes in the antigenic profile of the virus, which carries profound implications for vaccine seed stock preparation against avian H9N2 influenza. This report illustrates that aerosolized respiratory transmission is not exclusive to current human H1, H2, and H3 influenza subtypes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Respiratory droplet transmission of H9N2 avian–human reassortant viruses. Ferrets were inoculated with 106 TCID50 of P10 ferret-adapted H9N2 virus (A and C) or 2RCP10:6M98 reassortant virus (E). Direct contact ferrets (A, C, and E) and respiratory droplet contact ferrets for P10 (B and D) and 2RCP10:6M98 (F) were introduced at 24 h p.i. and nasal washes were collected daily. Black and white bars represent individual ferrets. In C, day 6 p.c., the direct contact from the group represented by the black bars died, as noted by an asterisk in the bar graph. Titers are expressed as log10 values of TCID50/mL with the limit of detection at 0.699 log10TCID50/mL.
Fig. 2.
Fig. 2.
Consistent isolation of P10 H9N2 virus in lung tissue. Two ferrets were infected with the ferret-adapted P10 virus or mock infected with PBS. Data are compared to the 2WF10:6M98 and WF10 viruses published in ref. . Tissues were collected at 5 dpi. *Note only 1 of 2 ferret lungs were positive for virus in the 2WF10:6M98 group. Titers are expressed as log10 values of TCID50/mL with the limit of detection at 0.699 log10TCID50/mL. OB, olfactory bulb; NT, nasal turbinate.
Fig. 3.
Fig. 3.
Adaptive mutations in the H9 HA surface protein necessary for respiratory droplet transmission. (A) Cartoon representation of the H9 HA monomer as described by Ha et al., (18) binding the Ltsc α2,6 sialic acid analog (orange and red lines) in the RBS. (B) Magnification of the globular head of the HA showing stick representations (in green) of key amino acids in the RBS: N183, G228, L226, T189, and V190, binding to α2,6 sialic acid (SIA, red lines). Numbers correspond to amino acid positions based on the H3 HA numbering system. (C) H9 HA RBS with amino acids corresponding to the WF10 HA wild-type sequence, which differs from the published crystal structure at 2 positions: H183 (dark blue stick) and E190 (red stick). T at position 189 is represented as a bright green stick. (D) H9 HA RBS with amino acids corresponding to the RCP10 HA sequence, which differs from the WF10 HA sequence at A189, represented as an olive green stick. Structures generated using MacPymol (DeLano Scientific).
Fig. 4.
Fig. 4.
Transmission phenotype supported through both T189A and G192R changes on the HA. Ferrets were inoculated intranasally (i.n.) with 106 TCID50 of either RCP10 (A189, G192) (A), RCP10 (T189, R192) (C), or 2WF10:6RCP10 virus (E). Twenty-four hours later, direct contact (A, C, and E) and respiratory droplet contact ferrets (B, D, and F) were introduced and nasal washes collected daily. Black and white bars represent individual ferrets. Titers are expressed as log10 values of TCID50/mL with the limit of detection at 0.699 log10TCID50/mL.
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