Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter, or stage. The Republic of Georgia is located at the intersection of three migratory flyways: the Central Asian flyway, the East Africa/West Asia flyway, and the Black Sea/Mediterranean flyway. For six complete study years (2010 to 2016), we collected AIV samples from various duck and gull species that breed, migrate, and overwinter in Georgia. We found a substantial subtype diversity of viruses that varied in prevalence from year to year. Low-pathogenic AIV (LPAIV) subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, and H16N3, and two highly pathogenic AIVs (HPAIVs) belonging to clade 2.3.4.4, H5N5 and H5N8, were found. Whole-genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide sequence diversity for LPAIVs among different host species. Hemagglutinin clade 2.3.4.4 H5N8 viruses, which circulated in Eurasia during 2014 and 2015, did not reassort, but analysis after their subsequent dissemination during 2016 and 2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions, with maintenance of local AIVs in Georgia, whereas other lineages showed considerable genetic interrelationships with viruses circulating in other parts of Eurasia and Africa, despite relative undersampling in the area. IMPORTANCE Waterbirds (e.g., gulls and ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate the dispersal of AIVs at intercontinental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or reassort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates AIV evolutionary dynamics within a multihost ecosystem at a stopover site where three major migratory flyways intersect. Our analysis of this ecosystem over a 6-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to mitigating both the risk of incursion into domestic poultry and the potential risk to mammalian hosts, including humans.

Craniofacial abnormalities often involve sutures, the growth centers of the skull. To characterize the organization and processes governing their development, we profile the murine frontal suture, a model for sutural growth and fusion, at the tissue- and single-cell level on embryonic days (E)16.5 and E18.5. For the wild-type suture, bulk RNA sequencing (RNA-seq) analysis identifies mesenchyme-, osteogenic front-, and stage-enriched genes and biological processes, as well as alternative splicing events modifying the extracellular matrix. Single-cell RNA-seq analysis distinguishes multiple subpopulations, of which five define a mesenchyme-osteoblast differentiation trajectory and show variation along the anteroposterior axis. Similar analyses of in vivo mouse models of impaired frontal suturogenesis in Saethre-Chotzen and Apert syndromes, Twist1+/- and Fgfr2+/S252W, demonstrate distinct transcriptional changes involving angiogenesis and ribogenesis, respectively. Co-expression network analysis reveals gene expression modules from which we validate key driver genes regulating osteoblast differentiation. Our study provides a global approach to gain insights into suturogenesis.

The relatively recent appearance of influenza A virus (IAV) epidemics in dogs expands our understanding of IAV host range and ecology, providing useful and relevant models for understanding critical factors involved in viral emergence. Here we integrate viral whole-genome sequence analysis and comprehensive surveillance data to examine the evolution of the emerging avian-origin H3N2 canine influenza virus (CIV), particularly the factors driving ongoing circulation and recent increases in incidence of the virus within the United States. Our results provide a detailed understanding of how H3N2 CIV achieves sustained circulation within the United States despite widespread host contact heterogeneity and recurrent epidemic fade-out. Moreover, our findings suggest that the types and intensities of selection pressures an emerging virus experiences are highly dependent on host population structure and ecology and may inhibit an emerging virus from acquiring sustained epidemic or pandemic circulation.

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.