In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses. IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

Previous studies have identified a functional role of PA-X for influenza viruses in mice and avian species; however, its role in swine remains unknown. Toward this, we constructed PA-X deficient virus (Sw-FS) in the background of a Triple-reassortment (TR) H1N2 swine influenza virus (SIV) to assess the impact of PA-X in viral virulence in pigs. Expression of PA-X in TR H1N2 SIV enhanced viral replication and host protein synthesis shutoff, and inhibited the mRNA levels of type I IFNs and proinflammatory cytokines in porcine cells. A delay of proinflammatory responses was observed in lungs of pigs infected by wild type SIV (Sw-WT) compared to Sw-FS. Furthermore, Sw-WT virus replicated and transmitted more efficiently than Sw-FS in pigs. These results highlight the importance of PA-X in the moderation of virulence and immune responses of TR SIV in swine, which indicated that PA-X is a pro-virulence factor in TR SIV in pigs.

A series of multiple logic circuits based on a single biomolecular platform is constructed to perform nonarithmetic and arithmetic functions, including 4-to-2 encoder, 1-to-2 demultiplexer, 1-to-4 demultiplexer, and multi-input OR gate. The encoder to a DNA circuit is the equivalent of a sensory receptor to a reflex arc. They all function to encode information from outside the pathway (DNA circuit or reflex arc) into a form that subsequent pathways can recognize and utilize. Current molecular encoders are based on optical or electrical signals as outputs, while DNA circuits are based on DNA strands as transmission signals. The output of existing encoders cannot be recognized by subsequent DNA circuits. It is the first time the DNA-based encoder with DNA strands as outputs can be truly applied to the DNA circuit, enabling the application of DNA circuits in non-binary biological environments. Another novel feature of the designed system is that the developed nanodevices all have a simple structure, low leakage and low crosstalk, which allows them to implement higher-level encoders and demultiplexers easily. Our work is based on the idea of complex functionality in a simple form, which will also provide a new route for developing advanced molecular logic circuits.

BackgroundH9N2 influenza viruses continuously circulate in multiple avian species and are repeatedly transmitted to humans, posing a significant threat to public health. To investigate the adaptation ability of H9N2 avian influenza viruses (AIVs) to mammals and the mutations related to the host switch events, we serially passaged in mice two H9N2 viruses of different HA lineages — A/Quail/Hong Kong/G1/97 (G1) of the G1-like lineage and A/chicken/Shandong/ZB/2007 (ZB) of the BJ/94-like lineage —and generated two mouse-adapted H9N2 viruses (G1-MA and ZB-MA) that possessed significantly higher virulence than the wide-type viruses.FindingZB-MA replicated systemically in mice. Genomic sequence alignment revealed 10 amino acid mutations coded by 4 different gene segments (PB2, PA, HA, and M) in G1-MA compared with the G1 virus and 23 amino acid mutations in 5 gene segments (PB1, PA, HA, M, and NS) in ZB-MA compared to ZB virus, indicating that the mutations in the polymerase, HA, M, and NS genes play critical roles in the adaptation of H9N2 AIVs to mammals, especially, the mutations of M1-Q198H and M1-A239T were shared in G1-MA and ZB-MA viruses. Additionally, several substitutions showed a higher frequency in human influenza viruses compared with avian viruses.ConclusionsDifferent lineages of H9N2 could adapt well in mice and some viruses could gain the ability to replicate systemically and become neurovirulent. Thus, it is essential to pay attention to the mammalian adaptive evolution of the H9N2 virus.

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