Huafen Zheng’s research while affiliated with Yangzhou University and other places

H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies. Supplementary Information The online version contains supplementary material available at 10.1186/s13567-024-01342-6.

Simple Summary Avian influenza viruses have the ability to breach species barriers and infect mammals, posing a significant threat to public health. The H7N9 subtype of avian influenza virus that emerged in China in 2013 resulted in 1568 human infections, with an alarming mortality rate of nearly 40%. While human infections caused by avian influenza viruses typically occur sporadically, the reasons behind the widespread impact of H7N9 remain unclear. During surveillance for avian influenza in live poultry markets in eastern China in 2013, we isolated two H7N9 avian influenza viruses from seemingly healthy chickens. One of these chicken-derived viruses demonstrated the ability to bind to mammalian receptors and naturally harbored the mammalian molecular marker PB2-627K. Furthermore, this virus proved to be highly pathogenic to mice. In summary, our findings suggest that the presence of a mammal-adapted H7N9 virus in poultry in 2013 could be a significant factor explaining the unusually high number of human infections during that period. Abstract Avian influenza viruses can cross species barriers and adapt to mammals. The H7N9 subtype AIV that emerged in China in 2013 caused 1568 human infections, with a mortality rate of nearly 40%. We conducted a retrospective analysis of H7N9 viruses that were isolated in live poultry markets in 2013. We found that two avian-origin H7N9 isolates, A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013, have a similar genetic background but exhibit different pathogenicity in mice. Whole-genome alignment of the two H7N9 viruses was carried out, and only six amino acid differences mapped in five genes, including the well-known virulence molecular marker PB2-E627K. Our retrospective analysis highlighted the importance of monitoring the adaptive mutations in avian influenza viruses with zoonotic potential.

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P + M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P + M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

Decades have passed since the first discovery of H10-subtype avian influenza virus (AIV) in chickens in 1949, and it has been detected in many species including mammals such as minks, pigs, seals and humans. Cases of human infections with H10N8 viruses identified in China in 2013 have raised widespread attention. Two novel reassortant H10N3 viruses were isolated from chickens in December 2019 in eastern China during routine surveillance for AIVs. The internal genes of these viruses were derived from genotype S (G57) H9N2 and were consistent with H5N6, H7N9 and H10N8, which cause fatal infections in humans. Their viral pathogenicity and transmissibility were further studied in different animal models. The two H10N3 isolates had low pathogenicity in chickens and were transmitted between chickens via direct contact. These viruses were highly pathogenic in mice and could be transmitted between guinea pigs via direct contact and respiratory droplets. More importantly, these viruses can bind to both human-type SAα-2,6-Gal receptors and avian-type SAα-2,3-Gal receptors. Asymptomatic shedding in chickens and good adaptability to mammals of these H10N3 isolates would make it easier to transmit to humans and pose a threat to public health.

Pigeons were previously thought to be resistant to H5 viruses and to play a minimal role in spreading these viruses. In this study, we evaluated the pathogenicity of two clade 2.3.4.4 H5N6 viruses in pigeons and the potential viral transmissibility to specific-pathogen-free chickens in direct close contact with experimentally infected pigeons. No pigeons from the A/goose/Eastern China/Xin/2015 (GS/Xin) group exhibited clinical signs or mortality, and the virus was only detected in a few organs. However, 3 of 12 pigeons inoculated with the A/goose/Eastern China/0326/2015 (GS/0326) virus died, and 7 of 12 showed neurological symptoms and efficient viral replication in multiple organs. In both groups, viral shedding occurred in only some of the pigeons, the shedding period was relatively short, and the infection was not transmitted to the chickens. We also used chicken, duck, and BALB/c mouse models to evaluate the pathogenicity of the two H5N6 isolates. Both H5N6 isolates showed highly pathogenic to chickens but different degrees of pathogenicity in mice. Interestingly, in ducks, the intravenous pathogenicity index indicated that the GS/Xin isolate was low pathogenic, and the GS/0326 isolate was highly pathogenic, corresponding to the pathogenicity in pigeons. Our results indicated that the pathogenicity of the clade 2.3.4.4 H5N6 virus is diverse in pigeons, and pigeons contribute little to its transmission among poultry. However, pigeons may still be potential healthy reservoirs of the H5N6 highly pathogenic avian influenza virus.