Anthony C. Fries

AftrRAD: a pipeline for accurate and efficient de novo assembly of RADseq data

An increase in studies using restriction site‐associated DNA sequencing (RADseq) methods has led to a need for both the development and assessment of novel bioinformatic tools that aid in the generation and analysis of these data. Here, we report the availability of AftrRAD, a bioinformatic pipeline that efficiently assembles and genotypes RADseq data, and outputs these data in various formats for downstream analyses. We use simulated and experimental data sets to evaluate AftrRADs ability to perform accurate de novo assembly of loci, and we compare its performance with two other commonly used programs, stacks and pyrad. We demonstrate that AftrRAD is able to accurately assemble loci, while accounting for indel variation among alleles, in a more computationally efficient manner than currently available programs. AftrRAD run times are not strongly affected by the number of samples in the data set, making this program a useful tool when multicore systems are not available for parallel processing, or when data sets include large numbers of samples.

Recovery of H14 influenza A virus isolates from sea ducks in the Western Hemisphere.

In 2010, H14 influenza A viruses were recovered from clinically normal sea ducks in the United States. These are the first H14 isolates recovered in the Western Hemisphere and represent the only documented H14 influenza A viruses isolated since the original isolates were recovered from near the Caspian Sea during 1982.

Genomic analyses detect Eurasian‐lineage H10 and additional H14 influenza A viruses recovered from waterfowl in the Central United States

The accurate and timely characterization of influenza A viruses (IAV) from natural reservoirs is essential for responses to animal and public health threats. Differences between antigenic and genetic subtyping results for 161 IAV isolates recovered from migratory birds in the central United States during 2010–2011 delayed the recognition of four isolates of interest. Genomic sequencing identified the first reported Eurasian‐origin H10 subtype in North America and three additional H14 isolates showing divergence from previously reported H14 isolates. Genomic analyses revealed additional diversity among IAV isolates not detected by antigenic subtyping and provided further insight into interhemispheric spread of avian‐origin IAVs.

Evidence for the circulation and inter-hemispheric movement of the H14 subtype influenza A virus.

Three H14 influenza A virus (IAV) isolates recovered in 2010 during routine virus surveillance along the Mississippi Migratory Bird Flyway in Wisconsin, U.S.A. raised questions about the natural history of these rare viruses. These were the first H14 IAV isolates recovered in the Western Hemisphere and the only H14 IAV isolates recovered since the original four isolates in 1982 in Asia. Full length genomic sequencing of the 2010 H14 isolates demonstrated the hemagglutinin (HA) gene from the 1982 and 2010 H14 isolates showed 89.6% nucleotide and 95.6% amino acid similarity and phylogenetic analysis of these viruses placed them with strong support within the H14 subtype lineage. The level of genomic divergence observed between the 1982 and 2010 viruses provides evidence that the H14 HA segment was circulating undetected in hosts and was not maintained in environmental stasis. Further, the evolutionary relationship observed between 1982 H14 and the closely related H4 subtype HA segments were similar to contemporary comparisons suggesting limited adaptive divergence between these sister subtypes. The nonstructural (NS) segment of one 2010 isolate was placed in a NS clade isolated infrequently over the last several decades that includes the NS segment from a previously reported 1982 H14 isolate indicating the existence of an unidentified pool of genomic diversity. An additional neuraminidase reassortment event indicated a recent inter-hemispheric gene flow from Asia into the center of North America. These results demonstrate temporal and spatial gaps in the understanding of IAV natural history. Additionally, the reassortment history of these viruses raises concern for the inter-continental spread of IAVs and the efficacy of current IAV surveillance efforts in detecting genomic diversity of viruses circulating in wild birds.

Spread and Persistence of Influenza A Viruses in Waterfowl Hosts in the North American Mississippi Migratory Flyway

ABSTRACT While geographic distance often restricts the spread of pathogens via hosts, this barrier may be compromised when host species are mobile. Migratory waterfowl in the order Anseriformes are important reservoir hosts for diverse populations of avian-origin influenza A viruses (AIVs) and are assumed to spread AIVs during their annual continental-scale migrations. However, support for this hypothesis is limited, and it is rarely tested using data from comprehensive surveillance efforts incorporating both the temporal and spatial aspects of host migratory patterns. We conducted intensive AIV surveillance of waterfowl using the North American Mississippi Migratory Flyway (MMF) over three autumn migratory seasons. Viral isolates (n = 297) from multiple host species were sequenced and analyzed for patterns of gene dispersal between northern staging and southern wintering locations. Using a phylogenetic and nucleotide identity framework, we observed a larger amount of gene dispersal within this flyway rather than between the other three longitudinally identified North American flyways. Across seasons, we observed patterns of regional persistence of diversity for each genomic segment, along with limited survival of dispersed AIV gene lineages. Reassortment increased with both time and distance, resulting in transient AIV constellations. This study shows that within the MMF, AIV gene flow favors spread along the migratory corridor within a season, and also that intensive surveillance during bird migration is important for identifying virus dispersal on time scales relevant to pandemic responsiveness. In addition, this study indicates that comprehensive monitoring programs to capture AIV diversity are critical for providing insight into AIV evolution and ecology in a major natural reservoir. IMPORTANCE Migratory birds are a reservoir for antigenic and genetic diversity of influenza A viruses (AIVs) and are implicated in the spread of virus diversity that has contributed to previous pandemic events. Evidence for dispersal of avian-origin AIVs by migratory birds is rarely examined on temporal scales relevant to pandemic or panzootic threats. Therefore, characterizing AIV movement by hosts within a migratory season is important for implementing effective surveillance strategies. We conducted surveillance following birds along a major North American migratory route and observed that within a migratory season, AIVs rapidly reassorted and gene lineages were dispersed primarily within the migratory corridor. Patterns of regional persistence were observed across seasons for each gene segment. We show that dispersal of AIV gene lineages by migratory birds occurs quickly along migratory routes and that surveillance for AIVs threatening human and animal health should focus attention on these routes.

Influenza A Viruses from Overwintering and Spring-Migrating Waterfowl in the Lake Erie Basin, United States.

SUMMARY. Influenza A virus (IAV) surveillance in migratory waterfowl in the United States has primarily occurred during late summer and the autumn southern migration. Data concerning the presence and ecology of IAVs in waterfowl during winter and spring seasons in the U.S. northern latitudes have been limited, mainly due to limited access to waterfowl for sampling. The southwestern Lake Erie Basin is an important stopover site for waterfowl during migration periods, and over the past 28 years, 8.72% of waterfowl sampled in this geographic location have been positive for IAV recovery during summer and autumn (June–December). To gain a better understanding of influenza A viral dynamics in waterfowl populations during winter and spring migration (February through April), cloacal swabs were collected from overwintering and spring-migrating waterfowl in Ohio and Michigan in 2006, 2007, 2013, and 2014. A total of 740 cloacal swabs were collected and tested using virus isolation in embryonating chicken eggs, resulting in the recovery of 33 (4.5%) IAV isolates. The influenza A isolates were recovered from eight waterfowl species in the order Anseriformes. Antigenically, the IAV isolates represent 15 distinct hemagglutinin (HA) and neuraminidase (NA) combinations, with seven (21%) of the isolates reported as mixed infections based on antigenic HA subtyping, NA subtyping, or both. This effort demonstrates the presence of antigenically diverse IAV in waterfowl during overwintering and spring migration at northern latitudes in the United States, thereby contributing to the understanding of the maintenance of diversity among waterfowl-origin IAVs.

assignPOP: An R package for population assignment using genetic, non-genetic, or integrated data in a machine-learning framework

Summary 1.The use of biomarkers (e.g., genetic, microchemical, and morphometric characteristics) to discriminate among and assign individuals to a population can benefit species conservation and management by facilitating our ability to understand population structure and demography. 2.Tools that can evaluate the reliability of large genomic datasets for population discrimination and assignment, as well as allow their integration with non-genetic markers for the same purpose, are lacking. Our R package, assignPOP, provides both functions in a supervised machine-learning framework. 3.assignPOP uses Monte-Carlo and K-fold cross-validation procedures, as well as principal component analysis (PCA), to estimate assignment accuracy and membership probabilities, using training (i.e., baseline source population) and test (i.e., validation) datasets that are independent. A user then can build a specified predictive model based on the relative sizes of these datasets and classification functions, including linear discriminant analysis, support vector machine, naive Bayes, decision tree, and random forest. 4.assignPOP can benefit any researcher who seeks to use genetic or non-genetic data to infer population structure and membership of individuals. assignPOP is a freely available R package under the GPL license, and can be downloaded from CRAN or at https://github.com/alexkychen/assignPOP. A comprehensive tutorial can also be found at https://alexkychen.github.io/assignPOP/. This article is protected by copyright. All rights reserved.