Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020-2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes.  Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021-2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities. IMPORTANCE High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.

Clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) from the 2021/22 epizootic is highly duck adapted and poorly adapted to chickens.

The 2021/2022 epizootic of high pathogenicity avian influenza (HPAIV) remains one of the largest ever in the UK, being caused by a clade 2.3.4.4b H5N1 HPAIV. This epizootic affected more than 145 poultry premises, most likely through independent incursion from infected wild birds, supported by more than 1700 individual detections of H5N1 from wild bird mortalities. Here an H5N1 HPAIV, representative of this epizootic (H5N1-21), was used to investigate its virulence, pathogenesis and transmission in layer chickens and Pekin ducks, two species of epidemiological importance. We inoculated both avian species with decreasing H5N1-21 doses. The virus was highly infectious in ducks, with high infection levels and accompanying shedding of viral RNA, even in ducks inoculated with the lowest dose, reflecting the strong waterfowl adaptation of the clade 2.3.4.4 HPAIVs. Duck-to-duck transmission was very efficient, coupled with high environmental contamination. H5N1-21 was frequently detected in water sources, serving as likely sources of infection for ducks, but inhalable dust and aerosols represented low transmission risks. In contrast, chickens inoculated with the highest dose exhibited lower rates of infection compared to ducks. There was no evidence for experimental H5N1-21 transmission to any naive chickens, in two stocking density scenarios, coupled with minimal and infrequent contamination being detected in the chicken environment. Systemic viral dissemination to multiple organs reflected the pathogenesis and high mortalities in both species. In summary, the H5N1-21 virus is highly infectious and transmissible in anseriformes, yet comparatively poorly adapted to galliformes, supporting strong host preferences for wild waterfowl. Key environmental matrices were also identified as being important in the epidemiological spread of this virus during the continuing epizootic.

Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b in Wild Birds and Poultry in Botswana.

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020-2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.

Intranasal Treatment of Ferrets with Inert Bacterial Spores Reduces Disease Caused by a Challenging H7N9 Avian Influenza Virus.

Background: Influenza is a respiratory infection that continues to present a major threat to human health, with ~500,000 deaths/year. Continued circulation of epidemic subtypes in humans and animals potentially increases the risk of future pandemics. Vaccination has failed to halt the evolution of this virus and next-generation prophylactic approaches are under development. Naked, "heat inactivated", or inert bacterial spores have been shown to protect against influenza in murine models. Methods: Ferrets were administered intranasal doses of inert bacterial spores (DSM 32444K) every 7 days for 4 weeks. Seven days after the last dose, the animals were challenged with avian H7N9 influenza A virus. Clinical signs of infection and viral shedding were monitored. Results: Clinical symptoms of infection were significantly reduced in animals dosed with DSM 32444K. The temporal kinetics of viral shedding was reduced but not prevented. Conclusion: Taken together, nasal dosing using heat-stable spores could provide a useful approach for influenza prophylaxis in both humans and animals.

c-Src-mediated phosphorylation of thyroid hormone receptor-interacting protein 6 (TRIP6) promotes osteoclast sealing zone formation.

Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647.

PURPOSE: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non-small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. EXPERIMENTAL DESIGN: The activity of EGFR inhibitors against WT and mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and T790M mutant EGFR) xenograft tumors. RESULTS: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. CONCLUSIONS: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.