PB1-F2 of Low Pathogenicity H7N7 Restricts Apoptosis in Avian Cells.

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs.

In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid "aa" in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Strain-dependent variations in replication of European clade 2.3.4.4b influenza A(H5N1) viruses in bovine cells and thermal inactivation in semi-skimmed or whole milk.

We investigated the thermostability of four European avian influenza A(H5N1) viruses in whole and semi-skimmed milk and their replication in bovine kidney and lung cells amid the current influenza A(H5N1) dairy cattle outbreak in the United States. Results showed strain-dependent differences in thermal inactivation, particularly in whole milk, and variable replication efficacy in lung cells. These findings support assessing the inactivation of European H5N1 viruses in milk and their replication in bovine cells, aiding biosafety protocols and public health measures.

Automated quantification of avian influenza virus antigen in different organs.

As immunohistochemistry is valuable for determining tissue and cell tropism of avian influenza viruses (AIV), but time-consuming, an artificial intelligence-based workflow was developed to automate the AIV antigen quantification. Organ samples from experimental AIV infections including brain, heart, lung and spleen on one slide, and liver and kidney on another slide were stained for influenza A-matrixprotein and analyzed with QuPath: Random trees algorithms were trained to identify the organs on each slide, followed by threshold-based quantification of the immunoreactive area. The algorithms were trained and tested on two different slide sets, then retrained on both and validated on a third set. Except for the kidney, the best algorithms for organ selection correctly identified the largest proportion of the organ area. For most organs, the immunoreactive area assessed following organ selection was significantly and positively correlated to a manually assessed semiquantitative score. In the validation set, intravenously infected chickens showed a generally higher percentage of immunoreactive area than chickens infected oculonasally. Variability between the slide sets and a similar tissue texture of some organs limited the ability of the algorithms to select certain organs. Generally, suitable correlations of the immunoreactivity data results were achieved, facilitating high-throughput analysis of AIV tissue tropism.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.