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Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

The identification of host factors involved in virus replication is important to understand virus life cycles better. Accordingly, we sought host factors that interact with the influenza viral nonstructural protein 2 by using coimmunoprecipitation followed by mass spectrometry. Among proteins associating with nonstructural protein 2, we focused on the β subunit of the F1Fo-ATPase, which received a high probability score in our mass spectrometry analysis. The siRNA-mediated down-regulation of the β subunit of the F1Fo-ATPase reduced influenza virion formation and virus growth in cell culture. We further found that efficient influenza virion formation requires the ATPase activity of F1Fo-ATPase and that plasma membrane-associated, but not mitochondrial, F1Fo-ATPase is important for influenza virion formation and budding. Hence, our data identify plasma membrane-associated F1Fo-ATPase as a critical host factor for efficient influenza virus replication.

We provide a brief introduction into the genome organization, life cycle, pathogenicity, and host range of influenza A viruses. We also briefly summarize influenza pandemics and currently available measures to control influenza virus outbreaks, including vaccines and antiviral compounds to influenza viruses.

The ability to modify influenza viruses at will has revolutionized influenza research. Reverse genetics has been used to generate mutant or reassortant influenza viruses to assess their replication, virulence, pathogenicity, host range, and transmissibility. Moreover, this technology is now being used to generate approved influenza virus vaccines and develop novel vaccines to combat seasonal and (future) pandemic influenza viruses. Several variations of the original system have been established, all of which are considerably robust and efficient.

Recent large-scale sequence analyses revealed 'signature' amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA and NP proteins of human influenza isolates ('human-like amino acids') in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

The spread of avian H5N1 influenza viruses around the globe has become a worldwide public health concern. To evaluate the pathogenic potential of reassortant viruses between currently cocirculating avian H5N1 and human H3N2 influenza viruses, we generated all the 254 combinations of reassortant viruses between A/chicken/South Kalimantan/UT6028/06 (SK06, H5N1) and A/Tokyo/Ut-Sk-1/07 (Tok07, H3N2) influenza viruses by reverse genetics. We found that the presence of Tok07 PB2 protein in the ribonucleoprotein (RNP) complex allowed efficient viral RNA transcription in a minigenome assay and that RNP activity played an essential role in the viability and replicative ability of the reassortant viruses. When the pathogenicity of 75 reassortant H5 viruses was tested in mice, 22 were more pathogenic than the parental SK06 virus, and three were extremely virulent. Strikingly, all 22 of these viruses obtained their PB2 segment from Tok07 virus. Further analysis showed that Tok07 PB1 alone lacked the ability to enhance the pathogenicity of the reassortant viruses but could do so by cooperating with Tok07 PB2. Our data demonstrate that reassortment between an avian H5N1 virus with low pathogenicity in mice and a human virus could result in highly pathogenic viruses and that the human virus PB2 segment functions in the background of an avian H5N1 virus, enhancing its virulence. Our findings highlight the importance of surveillance programs to monitor the emergence of human H5 reassortant viruses, especially those containing a PB2 segment of human origin.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.