Antigenic characterization of H3 and H7 avian influenza A virus from migratory waterfowl in North America

Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts including avian, swine, equine, canine, and sea mammals. These H3 viruses are both antigenically and genetically diverse. Here we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 1999 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other, and, to a lesser extent with those to H3 canine IAVs and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about 4 units, and each unit corresponds to a 2log2 difference in the HI titer. However, none of the tested H3 AIVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that these H3 avian IAVs we tested lack significant antigenic diversity, and these viruses are antigenically different from those viruses circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable. 12 Introduction The influenza A virus (IAV) causes a pandemic disaster impacting human health on multiple continents and then persist causing seasonal influenza epidemics [71, 72]. Four documented influenza A pandemics occurred in the last hundred years, 1918, 1957, 1968, and 2009. About 50 million people died of the 1918 H1N1 influenza A pandemic [73, 74]. The annual inter-pandemic influenza season in the northern hemisphere, from October to April, results in over 200,000 hospitalizations and up to 49,000 deaths annually in the United States [75-77]. IAVs have been recovered from at least 105 wild bird species from 26 different families [78, 79]. Among these birds, those living in wetland and aquatic environments (e.g., Anseriformes spp., particularly ducks, geese, and swans, and Charadriiformes spp., particularly gulls, terns, and waders) are by far the major source of these isolates and are generally accepted as a major natural IAV reservoir [24, 80]. Infected wild birds, especially migratory waterfowl, are believed to serve as a bridge facilitating the spread of IAVs between different wild avian species and different geographic locations, and are one source for viral transmission into domestic poultry [80, 81], swine [82], and humans [83]. The IAV has 8 genomic segments (segment 1-8) encoding for at least 11 proteins: PB2 by segment 1, PB1 and PB1-F2 by segment 2, PA by segment 3, hemagglutinin (HA) by segment 4, nucleoprotein (NP) by segment 5, neuraminidase (NA) by segment 6, matrix proteins M1 and M2 by segment 7, and nonstructural protein NS1 and NS2 by segment 8. To date, 18 HA and 11 NA subtypes have been reported in IAVs [84] although no isolates have been recovered for H17 and H18 subtypes. Antigenic changes 13 in the HA and NA, the two surface glycoproteins, are mainly due to accumulating point mutations resulting in antigenic drift or genomic reassortment resulting in antigenic shift. These changes in the HA are especially important as they allow an IAV to evade the herd immunity established from previous influenza infections or vaccination [21]. The H3 IAVs have very diverse host ranges in addition to infecting many species of birds they are widely found in mammals. After the emergence of the A/Hong Kong/1968 (H3N2) and 1968 pandemic this strain of IAV became endemic after the first year and has since been causing yearly seasonal epidemics in humans. The H3 IAVs maintained in infect swine, equine, canine, and avian species and cause sporadic outbreaks in sea mammals. H3N2 is one of the predominant IAVs infecting both domestic swine and feral swine [85-87]; H3N8 was shown to infect seals [88]; H3N8 is endemic in domestic canine in North America and equine worldwide [89, 90]; and the avian origin H3N2 infected domestic dogs in Asia [91, 92]. Previous studies documented substantial antigenic diversity in the H3 IAV in mammals [87]. Since 1968, the extent of antigenic drift has led to more than 28 updates of the H3N2 components in the seasonal influenza vaccines [93, 94]. In swine, the H3N2 IAVs are genetically and antigenically diverse. Four genetic clusters of H3N2 IAVs (clusters I–IV) have been identified in United States swine populations [82, 94-96], and the viruses in these four H3N2 clusters are antigenically different from each other. In addition, Cluster IV, which has become predominant among the United States swine population, has further evolved into two antigenic clusters: H3N2SIV-α and H3N2SIV-β [87]. Additionally, both H3N2 and H3N8 canine IAVs are antigenically distinct from contemporary human influenza viruses [97]. 14 In wild migratory birds, H3 is one of the more frequent hemagglutinin subtypes recovered in IAV surveillance projects [33]. However, the antigenic diversity of these H3 viruses is not well characterized. This study aims to characterize the antigenic diversity of contemporary H3 IAVs recovered from migratory birds in North America. This information helps us understand the natural history of influenza evolution and may also help improve knowledge on influenza prevention and control. Materials and Methods Viruses A total of 37 H3 isolates (H3N1, H3N2, H3N6, H3N7, H3N8, or H3N9) recovered from migratory birds in North America from 2007 to 2011 were included in this study (Table A.2). These viruses were recovered from American black duck (Anas rubripes), mallard (Anas platyrhynchos), ring-necked duck (Aythya collaris), hooded merganser (Lophodytes cucullatus), northern pintail (Anas acuta), snow goose (Chen caerulescens), blue-winged teal (Anas discors), and long-tailed duck (Clangula hyemalis). These H3 isolates were recovered from samples collected in Canadian locations in Nova Scotia, New Brunswick, Nunavut, and Prince Edward Island, as well as the US states of Maine, New Hampshire, North Dakota, Colorado, Oregon, Washington, Wisconsin, Iowa, Maryland, and New York, encompassing regions across the Atlantic, Mississippi, Central, and Pacific Bird Migratory Flyways in North America. In addition, one H3N8 isolate from a harbor seal (Phoca vitulina), A/harbor seal/New Hampshire/2011 (H3N8), was also included in this study. These viruses were recovered and propagated in 9-11 day-old specific pathogen free (SPF) embryonated chicken eggs before serological characterization. 15 Sera A total of 37 H3 isolates (H3N1, H3N2, H3N6, H3N7, H3N8, or H3N9) recovered from migratory birds in North America from 2007 to 2011 were included in this study (Table 1). These viruses were recovered from American black duck (Anas rubripes), mallard (Anas platyrhynchos), ring-necked duck (Aythya collaris), hooded merganser (Lophodytes cucullatus), northern pintail (Anas acuta), snow goose (Chen caerulescens), blue-winged teal (Anas discors), and long-tailed duck (Clangula hyemalis). These H3 isolates were recovered from samples collected in Canadian locations in Nova Scotia, New Brunswick, Nunavut, and Prince Edward Island, as well as the US states of Maine, New Hampshire, North Dakota, Colorado, Oregon, Washington, Wisconsin, Iowa, Maryland, and New York, encompassing regions across the Atlantic, Mississippi, Central, and Pacific Bird Migratory Flyways in North America. In addition, one H3N8 isolate from a harbor seal (Phoca vitulina), A/harbor seal/New Hampshire/2011 (H3N8), was also included in this study. These viruses were recovered and propagated in 9-11 day-old specific pathogen free (SPF) embryonated chicken eggs before serological characterization. Hemagglutination assay (HA) and hemagglutination inhibition assay (HI) The HA and HI assays were performed according to OIE guidelines [98] using 1% chicken red blood cells (Lampire Labs, Pipersville, PA).