Transboundary determinants of avian zoonotic infectious diseases: challenges for strengthening research capacity and connecting surveillance networks

As the climate changes, global systems have become increasingly unstable and unpredictable. This is particularly true for many disease systems, including subtypes of highly pathogenic avian influenzas (HPAIs) that are circulating the world. Ecological patterns once thought stable are changing, bringing new populations and organisms into contact with one another. Wild birds continue to be hosts and reservoirs for numerous zoonotic pathogens, and strains of HPAI and other pathogens have been introduced into new regions via migrating birds and transboundary trade of wild birds. With these expanding environmental changes, it is even more crucial that regions or counties that previously did not have surveillance programs develop the appropriate skills to sample wild birds and add to the understanding of pathogens in migratory and breeding birds through research. For example, little is known about wild bird infectious diseases and migration along the Mediterranean and Black Sea Flyway (MBSF), which connects Europe, Asia, and Africa. Focusing on avian influenza and the microbiome in migratory wild birds along the MBSF, this project seeks to understand the determinants of transboundary disease propagation and coinfection in regions that are connected by this flyway. Through the creation of a threat reduction network for avian diseases (Avian Zoonotic Disease Network, AZDN) in three countries along the MBSF (Georgia, Ukraine, and Jordan), this project is strengthening capacities for disease diagnostics; microbiomes; ecoimmunology; field biosafety; proper wildlife capture and handling; experimental design; statistical analysis; and vector sampling and biology. Here, we cover what is required to build a wild bird infectious disease research and surveillance program, which includes learning skills in proper bird capture and handling; biosafety and biosecurity; permits; next generation sequencing; leading-edge bioinformatics and statistical analyses; and vector and environmental sampling. Creating connected networks for avian influenzas and other pathogen surveillance will increase coordination and strengthen biosurveillance globally in wild birds

Avian Influenza Viruses in Wild Birds: Virus Evolution in a Multihost Ecosystem.

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

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Peer reviewed: TrueEmerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia