High genetic compatibility and increased pathogenicity of reassortants derived from avian H9N2 and pandemic H1N1/2009 influenza viruses

Abstract

H9N2 influenza viruses have been circulating worldwide in multiple avian species and repeatedly infecting mammals, including pigs and humans, posing a significant threat to public health. The coexistence of H9N2 and pandemic influenza H1N1/2009 viruses in pigs and humans provides an opportunity for these viruses to reassort. To evaluate the potential public risk of the reassortant viruses derived from these viruses, we used reverse genetics to generate 127 H9 reassortants derived from an avian H9N2 and a pandemic H1N1 virus, and evaluated their compatibility, replication ability, and virulence in mice. These hybrid viruses showed high genetic compatibility and more than half replicated to a high titer in vitro. In vivo studies of 73 of 127 reassortants revealed that all viruses were able to infect mice without prior adaptation and 8 reassortants exhibited higher pathogenicity than both parental viruses. All reassortants with higher virulence than parental viruses contained the PA gene from the 2009 pandemic virus, revealing the important role of the PA gene from the H1N1/2009 virus in generating a reassortant virus with high public health risk. Analyses of the polymerase activity of the 16 ribonucleoprotein combinations in vitro suggested that the PA of H1N1/2009 origin also enhanced polymerase activity. Our results indicate that some avian H9-pandemic reassortants could emerge with a potentially higher threat for humans and also highlight the importance of monitoring the H9-pandemic reassortant viruses that may arise, especially those that possess the PA gene of H1N1/2009 origin.

Structural basis for preferential avian receptor binding by the human-infecting H10N8 avian influenza virus

“…The rescued viruses were named rAH-H7N9 and rSH-H7N9, respectively, to distinguish from the natural isolates. A re-assortant JD-H10N8 (rJD-H10N8) virus was also rescued by plasmid-based reverse genetics technology 43 . The rJD-H10N8 virus comprised H10 HA and N8 NA from JD-H10N8 virus, and the remaining six gene segments are derived from PR8/H1N1 virus.…”

PA-X is a virulence factor in avian H9N2 influenza virus

“…A/chicken/Hebei/LC/2008 (HB/08, H9N2) virus was isolated from a diseased chicken in Hebei Province, China, in January 2008 and propagated in 10-day-old specific pathogen-free embryonated chicken eggs (Sun et al, 2011). 293T, MDCK and A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10 % FBS (Life Technologies), 100 U penicillin ml 21 and 100 g streptomycin ml 21 .…”

“…All eight gene segments were previously amplified by reverse transcription (RT)-PCR from HB/08 virus and cloned into the dual-promoter plasmid pHW2000 (Sun et al, 2011). PA-X-deficient virus H9N2-FS was created by site-directed mutagenesis (QuikChange mutagenesis kit; Agilent) on the corresponding PA gene of H9N2 WT virus, which converted the frameshifting motif from UCC UUU CGU to AGC UUC AGA (U592A, C593G, U597C, C598A and U600A) to prevent the formation of PA-X (Jagger et al, 2012).…”

“…In the present study, the use of reverse genetics was based on the A/chicken/Hebei/LC/2008 (H9N2 WT) virus (Sun et al, 2011). To evaluate the effect of loss of PA-X expression on viral function, we generated PA-X-deficient virus H9N2-FS by altering the frameshifting motif from UCC UUU CGU to AGC UUC AGA in the PA segment to prevent the formation of PA-X (Fig.…”

Virulence of pandemic (H1N1) 2009 influenza A polymerase reassortant viruses

“…In experimental settings, several indications on the potential pathogenic impact or contribution of the viral segments derived from the pandemic virus toward contemporary viruses have been observed. Acquisition of the pandemic PA gene increased pathogenicity of an avian H9N2 (A/Chicken/Hebei/LC/2008) virus in mice 30 whereas reassortants containing one or both of the PB2 and PB1 subunits from CA04 induced enhanced growth kinetics of a human HPAI H5N1 (A/Vietnam/HN31604/2009) isolate. 24 Therefore, the pandemic (H1N1) 2009 virus may also become a significant genetic reservoir, a source for the generation of novel viruses with potential threat to public health.…”