A/Chicken/Beijing/1/94 (Ck/BJ/1/94) avian influenza virus (AIV), a prototype of the H9N2 subtype, is phylogenetically similar in its hemagglutinin (HA) and neuraminidase (NA) genes to A/Chicken/Shanghai/F/98 (Ck/SH/F/98; H9N2) AIV, a natural reassortant between different sublineages. To understand the role of HA and NA genes in the airborne transmission of H9N2 AIV, we compared the transmission route and the relative replication efficiency of these strains in specific-pathogen-free chickens. Three recombinant viruses were generated by reverse genetics, containing the HA and NA genes (or both) from A/Chicken/Guangdong/SS/94 (Ck/GD/SS/94), in a background of internal genes derived from Ck/SH/F/98. Inoculated chickens were kept in either direct or indirect contact with uninoculated chickens, and viral shedding and titers were monitored. The results showed that Ck/GD/SS/94 lacks the ability to be transmitted through indirect contact, while Ck/SH/F/98 could be transmitted indirectly. Recombinant virus (RF/SSHA), containing the internal genes of Ck/SH/F/98 and the HA gene of Ck/GD/SS/94, resulted in decreased viral titers in lung tissue as compared to the parental strain. Interestingly, substituting the NA gene, or both the NA and HA genes, of Ck/SH/F/98 with that of Ck/GD/SS/94 completely abolished the airborne transmission of the recombinant RF/SSNA and RF/SSHA/SSNA. In conclusion, Ck/SH/F/98 acquired the ability of airborne transmission and replicated with a higher efficiency in the respiratory tract of the chickens. Our data indicated that the NA gene of Ck/SH/F/98 can affect virus replication and, therefore, indirectly affect the transmission for the gene constellations of these viruses.

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