32Wild aquatic birds maintain a large genetically diverse pool of influenza A viruses (IAVs), which can be 33 transmitted to lower mammals and ultimately humans. Through phenotypic analyses, only a small set of 34 avian IAVs replicated well in the epithelial cells of swine upper respiratory tracts, and these viruses were 35 shown to infect and cause virus shedding in pigs. Such a phenotypic trait appears to emerge randomly and 36 are distributed among IAVs across multiple avian species, geographic and temporal orders, and is 37 determined not by receptor binding preference but other markers across genomic segments, such as those 38 in the ribonucleoprotein complex. This study demonstrates that phenotypic variants exist among avian 39 IAVs, only a few of which may result in viral shedding in pigs upon infection, providing opportunities for 40 these viruses to become pig adapted, thus posing a higher potential risk for creating novel variants or 41 detrimental reassortants within pig populations. 42 43 44 45 46 47 Author Summary 49Having both avian-like receptors and human-like α2,6-linked sialic acid receptors, swine serve as a 50 "mixing vessel" for generating human influenza pandemic strains. All HA subtypes of IAVs can infect 51 swine; however, only sporadic cases of avian IAVs are reported in domestic swine. The molecular 52 mechanisms affecting avian IAVs ability to infect swine are still not fully understood. Through 53 phenotypic analyses, this study suggested that tissue tropisms (i.e., in swine upper respiratory tracts) of 54 avian IAVs affect their spillovers from wild birds to pigs, and this phenotype was determined not by 55 receptor binding preference but by other markers across genomic segments, such as those in the 56 ribonucleoprotein complex. In addition, our results showed that such a phenotypic trait was sporadically 57 and randomly distributed among IAVs across multiple avian species, geographic and temporal orders. 58This study suggested an efficient way for risk assessment of avian IAVs, such as in evaluating their 59 potentials to be transmitted from avian to pigs. 60 61 Influenza A viruses (IAVs), a member in the Orthomyxoviridae family, are a negative single stranded 63 RNA virus with eight gene segments, encoding at least 11 proteins. The nomenclatures of IAVs are 64 determined based on antigenic properties of two surface glycoproteins, Hemagglutinin (HA) and 65 Neuraminidase (NA). To date, 18 HA subtypes and 11 NA subtypes have been identified [1, 2]. Besides 66 humans, pigs, dogs, and horses, IAVs have been recovered from variety of bird species, including at least 67 105 wild bird species of 26 different families [3]. Among wild birds, birds of wetlands and aquatic 68 environments such as the Anseriformes (particularly ducks, geese, and swans) and Charadriiformes 69 (particularly gulls, terns, and waders) constitute the major natural IAV reservoir [4]. They maintain a 70 large IAV genetic pool, which contributes to the appearance of new IAVs in other birds, lower mammals, 71 and ultima...